Centrifuge

ABSTRACT

Centrifuges are useful to, among other things, remove red blood cells from whole blood and retain platelets and other factors in a reduced volume of plasma. Platelet rich plasma (PRP) and or platelet poor plasma (PPP) can be obtained rapidly and is ready for immediate injection into the host. Embodiments may include valves, operated manually or automatically, to open ports that discharge the excess red blood cells and the excess plasma into separate receivers while retaining the platelets and other factors in the centrifuge chamber. High speeds used allow simple and small embodiments to be used at the patient&#39;s side during surgical procedures. The embodiments can also be used for the separation of liquids or slurries in other fields such as, for example, the separation of pigments or lubricants.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is divisional of currently U.S. patent application Ser.No. 14/488,556, filed on Sep. 17, 2014, which is a Continuation of

U.S. patent application Ser. No. 13/766,528, filed on Feb. 13, 2013, nowU.S. Pat. No. 8,870,733, which is a Continuation-In-Part of

U.S. patent application Ser. No. 13/447,008, filed on Apr. 13, 2012, nowU.S. Pat. No. 8,394,006, which is a Continuation-In-Part of

U.S. patent application Ser. No. 13/396,600, filed on Feb. 15, 2012, nowU.S. Pat. No. 8,556,794, which is a Continuation-In-Part of

U.S. patent application Ser. No. 13/209,226, filed on Aug. 12, 2011, nowU.S. Pat. No. 8,469,871, which is a Continuation-In-Part of

U.S. patent application Ser. No. 12/949,781, filed on Nov. 19, 2010, nowU.S. Pat. No. 8,317,672.

The above mentioned Ser. No. 13/396,600 application is also aContinuation-In-Part of now expired PCT International Patent ApplicationS.N. PCT/US11/01922, filed on Nov. 19, 2011, and designating the U.S.,which is a Continuation-In-Part of the above mentioned U.S. patentapplication Ser. No. 13/209,226, filed on Aug. 12, 2011, now U.S. Pat.No. 8,469,871, and is a Continuation-In-Part of the above mentioned U.S.patent application Ser. No. 12/949,781, filed on Nov. 19, 2010, now U.S.Pat. No. 8,317,672.

All of the above listed applications are assigned to the same assigneeas this invention and whose disclosures are incorporated by referenceherein.

TECHNICAL FIELD

The present invention pertains to centrifuges.

BACKGROUND ART

Fluids, such as whole blood or various other biological fluids aresuspensions and can be separated into their constituent parts orfractions. For example, whole blood comprises four main fractions, redblood cells, white blood cells, platelets and plasma, that can beseparated based on their different specific gravities in a device suchas a centrifuge. An anti-coagulated whole blood sample may be placed ina test tube, or other similar device, which is then spun in a centrifugeat a specified speed. The generated centrifugal force separates theblood into the different fractions based on their relative specificgravities. The red blood cells are on the bottom, plasma, is on the topwith the intermediate specific gravity white blood cells and platelets(together referred to as the buffy coat (BC)) intermediate to the othertwo fractions. Various other biological fluids may be separated as well.For example, nucleated cells may be separated and extracted from bonemarrow or adipose tissue derived samples.

It is desirable to isolate the different fractions of whole blood fordiffering medicinal purposes. The platelets can be obtained inpreparations of platelet rich plasma (PRP) or platelet concentrates(PC). Platelets contain growth factors (e.g. PDGF, TGF-β, and others),which may initiate, aid in or accelerate various bodily functions,including but not limited to angiogenesis, wound healing, andosteogenesis. Administering autologous platelets to an injury site mayimprove the healing response by using a patient's own platelets withoutthe risk of infection by using blood products from another donor source.Alternatively, platelet poor plasma (PPP) may be desired for use invarious procedures. PPP may be prepared by isolating the plasma fractionfrom platelet concentrates, and preserving the isolated plasma fraction.

Various systems exist for the production of PRP/PC. Some use specializedtest tubes, U.S. Pat. Nos. 7,179,391 and 7,520,402, that can includefloats, tubing and/or gel materials of specific densities. Other systemsuse specialized double syringes, for example those found in U.S. Pat.Nos. 6,716,187 and 7,195,606. These test tubes and syringes must becentrifuged in a specialized large centrifuge for a specified time,typically 10-30 minutes, and then by delicate handling and extraction ordecanting procedures produce the desired PRP/PC. The consistency ofthese preparations can vary depending on the operator's skill level.Other systems, for example U.S. Pat. No. 6,982,038, contain specializedcentrifuge chambers and complicated control systems to produce thePRP/PC in about 30 minutes. All of these systems provide PRP/PC ofdiffering platelet concentrations depending on the method used. A majordrawback to these methods is the need for an expensive piece of capitalequipment which limits the utility to facilities that have the funds andspace available. These methods also require considerable operator skillsto complete the procedures necessary to obtain the PRP/PC.

The ability to produce PRP/PC from a patient's own blood at the point ofcare without the need for complex, expensive equipment and difficultprocedures would facilitate the clinical utility of PRP/PC. Thereforethe objects of this invention include among other things providing anapparatus and method for processing a patient's own blood at the pointof care in a short period of time that is self-contained, batteryoperated, small and or portable, inexpensive, easy to use, reproducible,able to separate many cellular populations, and disposable without theneed for additional centrifugation equipment.

DISCLOSURE OF THE INVENTION

In accordance with the invention, a single use, sterile, self-contained,compact, easy to use centrifugal separation unit provides for quick,reliable concentration of constituents of a liquid mixture, for example,a biologic liquid mixture, such as platelet concentration from wholeblood, or alternatively concentrating cells from bone marrow aspirate.The resultant PRP/PC can be immediately used for application to thepatient. The unit is suitable for office, operating room, emergency use,or military field hospital use.

The disposable self-contained PRP separator features a motor with adrive axis, the drive axis being coaxial with the central orlongitudinal axis of the blood separation chamber (BSC) assembly. Themotor can have the capacity to rotate the BSC at speeds in the range10,000 to 25,000 RPM for several minutes. Power can be supplied to themotor through a battery or other power pack. The power can be connectedthrough a switch and even small dry cell batteries will have sufficientcapacity to complete the separation process. The BSC and motor/batteryare fully enclosed in an outer container that includes an access port tothe BSC to which a standard syringe can be attached. Alternatively theBSC can be rotated by non-electrical means such as an air driven turbineor spring drive. It could also include a magnetic or mechanical couplingto an external drive motor, or any source of energy that may beavailable at the surgical site for example in the surgical suite or onlocation during a trauma procedure, such as at a “MASH” compound.

In a first embodiment the BSC assembly features a barrel that may becylindrical or tapered, an end cap incorporating passageways and atubular extension, and in some embodiments a piston or bladder, thatbetween them define the BSC. A sleeve sliding over the outer diameter ofthe end cap acts as the moving part of two valve assemblies, each valvefeaturing a recess in the outer surface of the end cap and an O-ring inthe recess. Passages within the end cap lead from the BSC to the recesscenters, and two ports in the sleeve align with the recess centers in a3 position sequence. The two ports in the sleeve are positioned so thatthey do not align with the two recess centers in the end cap at the sametime. In sequence, the sleeve selects a first port open, then both portsclosed, and then a second port open. The ports are opened in a stepwisemotion, but could be opened proportionally. The sleeve is operated by aknob connected to a slidable collar through a bearing assembly so thatthe knob does not rotate during operation of the motor.

Anti-coagulated blood is injected through the tubular extension in orderto fill the BSC. The sleeve is in a first position where both ports onthe sleeve do not align with either of the recesses in the end cap. Themotor is actuated and the BSC rotates to create a centrifugal force onthe blood thereby separating it into its components with the red bloodcells closest to the inner wall of the BSC with the white blood cellslining the red blood cell layer toward the center, followed by theplatelets and then plasma filling the center. In other words, thecentrifugation yields concentric stratified constituent layers of themixture, with adjacent concentric stratified constituent layers defininga mixture interface. After a centrifugation period of about 1 minute orless the sleeve is moved to a second position in which the first port inthe sleeve aligns with the recess in the end cap. This port communicateswith the layer of red blood cells against the inner wall. The red bloodcells will exit the chamber through this port due to pressure generatedby the centrifugal force. As red blood cells exit the separator, thevolume is replaced by air entering through the tubular extension in theend cap. The air forms a column in the center of the chamber that growslarger as more volume is replaced. It is also conceived that without anair inlet vent, that continued rotation and evacuation of the red bloodcells will result in a vacuum core being formed, as the blood isdegassed and possibly drawing vapor from the liquid due to the reducedpressure at the center of rotation. After a substantial amount,preferably the majority, of the red blood cells are discharged from theblood separator volume, the sleeve is moved to a third position to closethe first port and open the second port. This is done before the layerof platelets in the volume can exit the first port. The passage to thesecond recess in the end cap of the device is precisely positioned awayfrom the center axis to remove a volume of plasma from the BSC withoutdisturbing the platelet layer. As plasma leaves the chamber, airreplaces the volume through the tubular extension and the column of airin the center of the BSC continues to grow in diameter. When thediameter of the air column encompasses the second passage entrance, nomore plasma can exit the chamber and the concentration process isthereby automatically ended. In the case where there is a vacuum corecreated, the concentration process would automatically end in a similarmanner, as the vacuum core encounters the second passage entrance. Thedevice is turned off and the platelet concentrate is ready for use.

Another embodiment uses a flexible bladder lining the interior of theBSC. The solid end of the BSC includes a hole for air to enter aroundthe exterior of the flexible bladder. The end cap axis tubular extensionincludes an airtight valve. This embodiment operates in the same mannerexcept that it does not deliberately introduce air into contact with theblood sample. During the centrifugation cycle while red blood cells andthen plasma are exiting the chamber, air enters the opposite side of thechamber thus collapsing the flexible bladder. Due to the pressuregenerated in the liquid by centrifugal force, the sack collapses into a“W” shape with the open ends of the “W” facing toward the end of thechamber opposite the end with the air bleed hole. As more plasma exitsthe chamber the middle of the “W” reaches the second passage in the endcap and closes the passage off thus automatically ending the cycle.

Another embodiment replaces the flexible bladder with a piston andspring: as red blood cells (RBCs) exit the valve ports, the piston movestowards the end cap encouraged by the spring.

It is further disclosed that the system of the subject invention mayincorporate an automatic shutoff mechanism to seal the port(s) basedupon certain conditions. For example, one such mechanism can incorporatea flowable separation aid in the form of a gel of an intermediatespecific gravity selected to be between an undesired element, e.g. redblood cells, and a desired therapeutic element, e.g. platelets. Theseparator gel viscosity is designed so that it will not pass through thesmall exit port at the centrifuge speed employed in the blood separationcentrifuge. Upon activation of the centrifuge, the separator gel wouldcreate a distinct layer and barrier between the outer red blood celllayer, located near the periphery of the axis of rotation, and theplatelet poor layer which would be located closer to the center axis ofthe centrifuge rotation. The separator gel automatically plugs the firstport when all of the red blood cells have exited. As a further example,the automatic shut-off of the first port can be accomplished with asolid damper, or vent flap, also constructed of a material with aspecifically targeted intermediate specific gravity. Upon initialoperation, the damper would open and separate away from the vent holebased upon its density and attempt to position itself at a locationbetween the red blood cells and the platelets. As in the previousexample, once the red blood cells have fully exited the system, thedamper would seal the vent hole and effectively prevent the plateletrich fluid from exited the system. As yet another example of aseparation aid, plastic beads such as microspheres with the desiredintermediate specific gravity could also be pre-located within thecentrifuge chamber. The beads would be sized appropriately to plug theexit port after the undesirable element, e.g. red blood cells, exitedthe system.

In another embodiment, the BSC, or at least a portion thereof, can bemade of a clear (transparent) material so that the progress of the redblood cell removal can be observed through a clear window in the outercase. This can allow for precise timing for closing the first port toend the exiting of the red blood cells.

Another embodiment accomplishes the concentration through precise timingof the valve opening/closing sequence and the starting and stopping ofthe motor.

In another embodiment, the system may feature a reusable drive componentwith a motor that is arranged to be coupled to a disposable centrifugecomponent, wherein the blood products are centrifuged, separated, andcontained entirely within the disposable unit, such that the drivecomponent is not exposed to blood product and may be reused without fearof contamination.

In another embodiment, the disposable unit may include blood absorbentmaterials or fluid receiving chambers to capture the evacuated bloodproducts.

In another embodiment, the rotation chamber is arranged to minimize thedisruption to the interfaces between the separated blood products, whilethe red blood cells and plasma components are evacuated from therotating chamber.

BRIEF DESCRIPTIONS OF THE DRAWINGS

FIGS. 1a and 1b : Principle of operation.

FIG. 2: Centrifuge with spring loaded piston in tapered chamber, chargeposition, RBC valve open, Plasma valve closed (Longitudinal partsection).

FIGS. 3a, 3b, 3c, and 3d show transverse sections of the centrifuge withspring loaded piston in tapered chamber, (transverse sections of FIG.2), and enlarged details of the RBC valve components used in all devicesshown in FIGS. 2, 4, 5, 6, 7, 9, 10, 11, 12, 14, 15, 16, 17, and 18.

FIG. 4: Centrifuge with spring-loaded piston in tapered chamber,spin-down, RBCs separated from plasma, both valves closed (Longitudinalpart section).

FIG. 5: Centrifuge with spring-loaded piston in tapered chamber, midposition, RBC valve open and RBCs being dumped, plasma valve closed(Longitudinal part section).

FIG. 6: Centrifuge with spring-loaded piston in tapered chamber, finalposition, RBC valve closed, plasma valve open and most of plasma dumped(Longitudinal part section).

FIG. 7: Centrifuge with bladder chamber, charge position, RBC valveopen, plasma valve closed (Longitudinal part section).

FIG. 8: Centrifuge with bladder chamber, charge position, (transversesection of FIG. 7).

FIG. 9: Centrifuge with bladder chamber, spin-down, RBCs separated fromplasma, both valves closed, (longitudinal part section).

FIG. 10: Centrifuge with bladder chamber, RBCs dumping position, RBCvalve open, plasma valve closed (Longitudinal part section).

FIG. 11: Centrifuge with bladder chamber, Plasma valve open, RBC valveclosed, plasma being dumped (Longitudinal part section).

FIG. 12: Centrifuge with air core, initial charge position, both valvesclosed. (Longitudinal part section).

FIG. 13: Centrifuge with air core, (transverse section of FIG. 12).

FIG. 14: Centrifuge with air core, spin and separate, RBCs being dumped,RBC valve open, plasma valve closed (Longitudinal part section).

FIG. 15: Centrifuge with air core, RBC valve closed, plasma valve open,residual RBCs and residual plasma remaining (Longitudinal part section).

FIG. 16: Centrifuge with air core, removal of PRP at finish, both valvesclosed (Longitudinal part section).

FIG. 17: Centrifuge with a typical enclosure (Longitudinal part section,showing RBC and plasma capture means and aerosol prevention means).

FIGS. 18a and 18b : Centrifuge with typical enclosure, (transversesection of FIG. 17).

FIG. 19. Simplified longitudinal cross section of centrifuge withdisposable and reusable components shown separated. Shown with the redblood cell and plasma valves closed.

FIG. 20a . Simplified schematic of centrifuge chamber having a plenum atthe end of the red blood cell channel and separated fluids.

FIG. 20b . Projection view of the plasma port of FIG. 20a with plasmafluid flow pattern represented by arrows.

FIG. 21. Assembled centrifuge in running position, RBC valve open andRBC dump complete.

FIG. 22. Simplified transverse section of FIG. 21 at AA.

FIG. 23. Simplified transverse section of FIG. 21 through plasma valveat BB showing valve construction.

FIG. 24. Assembled centrifuge in running position, RBC valve shut,plasma valve open and plasma dump complete.

FIG. 25. Centrifuge with means for gathering Platelet Poor Plasma (PPP)in a separate receiver, shown in plasma collection phase of operation.

FIG. 26. Centrifuge with absorbent washers to capture blood products,shown at the end of the RBC dump phase.

FIG. 27a . Simplified schematic of centrifuge chamber having plena atthe end of the red blood cell channel and at the plasma outlet, andseparated fluids.

FIG. 27b . Projection view of the plasma port of FIG. 27A, with fluidflow pattern represented by arrows.

FIG. 28. Cross section views of alternate RBC-Plasma receiver withindexing valve, depicted in the closed position.

FIG. 29. Cross section view of alternate RBC-Plasma receiver withindexing valve showing valve in open position.

FIG. 30. Isometric cross section view of alternate RBC-Plasma receiverwith indexing valve.

FIG. 31. Isometric view of disassembled alternate RBC-Plasma receiverwith indexing valve.

FIG. 32. Centrifuge with scaffold material in a compartment and arrangedfor receipt of buffy coat component.

FIG. 33. Simplified schematic of centrifuge chamber having a restrictionfeature in a portion of the circumferential channel and separatedfluids.

FIG. 33a . Transverse sectional view of section A-A of FIG. 33, with theportion of the circumferential channel corresponding to angle dfeaturing a restriction feature.

FIG. 34 Cross-sectional view of another alternative centrifuge with adisk-shaped rotating assembly.

FIG. 35 Enlarged cross-sectional view the details of the valvearrangement of the disk-shaped rotating assembly of the centrifuge ofFIG. 34.

FIG. 36 Enlarged, exploded isometric view of components of the forcetransfer mechanism of the centrifuge of FIG. 34.

FIG. 37 Enlarged projection view of the valve components of the rotatingassembly of the centrifuge of FIG. 34.

MODES FOR CARRYING OUT THE INVENTION

FIG. 1a provides an illustration for description of the principle ofoperation of the devices covered in this invention. A chamber ofessentially frusto-conical shape 1, contains a mixture of severalliquids of differing densities, and rotates about the longitudinal axisXX. The liquids 2, 3, and 4 separate into radially distinct layers asshown in section AA. The taper is beneficial in several ways, first itallows a small volume of liquid to offer a large radial depth (as shownat 11) compared with the radial depth the same volume would have ifdistributed over the whole length of a right circular cylinder ofsimilar dimensions, see FIG. 1b at 14. Second, the taper provides acomponent of radial acceleration force that helps to scour the outerliquid constituent towards a port 9 placed at the larger cone diameter.Third, the taper also allows visualization of the constituent boundariesas axial locations such as 5 and 6 instead of radial locations such as 7and 8 in some of the embodiments. It should be pointed out at thisjuncture that the term “taper” or “tapered” is used in its normaldefinitional sense, i.e., to become progressively smaller toward one endor to diminish gradually. Thus the taper of the chamber need not belinear, as shown in the exemplary embodiments contained herein, but maybe arcuate or of other shapes as set forth herein. In severalembodiments the wall 12 of FIG. 1 moves toward the larger diameter andthe frusto-conical volume reduces as one or more constituents are portedfrom the ports, for example at 9 and 10, leaving the center constituent3 at its original volume. In other embodiments wall 12 remains in placeand air is introduced on the center line at 13 to permit the porting ofconstituents 2 and 4 at 9 and 10 as the air core expands to replace thedischarged constituents.

FIG. 2 is a mainly longitudinal section of an essentially circulardevice, external housing not shown. In FIG. 2 a liquid tight variablevolume, the chamber (BSC), is formed from a tapered barrel 206, piston210, piston seal 211 and end cap 215. Piston 210 and seal 211 are biasedtoward the larger end of the BSC by spring 209. Larger end of barrel 206is closed by end cap 215. The inner surface of the end cap 215 forms thelarger diameter end wall of the chamber, with the inner surface of thebarrel 206 forming the chamber's tapering side wall. In the case wherethis device is used to enrich plasma from whole blood, end cap 215 haspassages 216 and 217 bored within to permit the passage of red bloodcells from passage 217 and plasma from passage 216. Passage 217 is shownpassing through the outside skirt of the end cap that is in line withthe outside wall of tapered barrel 206. A passage bored 90° from thatshown at 217; through the inside face of end cap 215 at the maximum IDposition would be functionally equivalent to the one shown at 217 andwould have a shape similar to passage 216. Passages 217 and 216 connectwith valves formed by O-rings 218 compressed in recesses 226 operatingin concert with ports 228 and 227 respectively in sleeve 213. Thesevalve components are shown enlarged in FIGS. 3b and 3d . Sleeve 213 fitsslidably on end cap 215 to permit the port holes 228 and 227 to connectwith the passages 216 and 217 at appropriate points in the operation.Sleeve 213 is keyed to end cap 215 to permit the transmission of rotarymotion between these constituents (key not shown). Insert 219 isfastened to end cap 215 to provide an axle for the ball bearing 220supporting the left hand end of the rotating assembly. Since the sleeve213 is rotating with the chamber, a ball bearing 221 is provide toconnect the sleeve to a non-revolving knob 223 via collar 225 and rods222. The knob and sleeve can be placed in 3 positions: first position,port 228 open and port 227 closed: second position, both ports 227 and228 closed: third position, port 228 closed and port 227 open. Barrel206 is fastened to the shaft 205 of electric motor 201 using screw 207.No additional bearings are provided at the motor end, the motor bearingssufficing to support the barrel. The complete assembly is supported by aframe 208, the insert bearing 220 and the motor 201 being located onthis same frame. The rotating components all rotate about axis XX.

To use the device for preparing PRP, a syringe 233 with needle 234,filled with anti-coagulated whole blood is inserted into the devicethrough elastomeric seal 214 to load the chamber with whole blood 229.Knob 223 is placed in the first position to allow air to discharge fromport 228 as the chamber is filled with blood. Whole blood 229 fullycharges the chamber pushing the piston 210 and seal 211 to the farright, compressing spring 209.

FIG. 3a , a cross section at AA in FIG. 2, clarifies the construction ofthe knob 223 and rod components 222. FIG. 3b is a cross section at BB inFIG. 2 showing details for the valve components, those being the recess226 in end cap 215, O-ring 218 and port 228 in sleeve 213 (theconstruction of the valve for port 227 is the same). FIG. 3c shows thesection at CC of FIG. 2.

Once the chamber has been charged with whole blood, the knob and sleeveare placed in the second position with both valves closed, the syringe223 is removed and the motor started. The motor is then run for timesbetween 15 and 90 seconds depending on the speed used. Speeds of 10,000rpm to 25,000 rpm have been used, developing centrifugal accelerationsat the outside of the spinning chamber from 1000 g to 6000 g.

FIG. 4 shows the device of FIG. 2 in operation rotating at speed. TheRBC port 228 and the plasma port 227 are both closed. The boundarybetween the RBC layer and the plasma layer is shown at 237. The piston210 is still at the as-charged position and the spring 209 is fullycompressed. The spring has two functions, it moves the piston to theleft as red blood cells are discharged from the chamber through port228, and the spring creates a significant minimum pressure in therevolving liquid: this prevents the core of the spinning liquid fromreaching the vapor pressure of the liquids and may suppress cell damagein some circumstances.

Once the red blood cells and the plasma have separated, with the devicestill rotating, the knob and sleeve are placed in the first position andred blood cells are discharged from port 228 into the casing (casing notshown, but see FIGS. 17 and 18) surrounding the device. FIG. 5 shows thesituation at the mid-point of the RBC 231 discharge when the piston 210is in mid position. Once the majority of red blood cells have beendischarged the valve is placed in the third position and plasma 230 iseliminated from port 227. FIG. 6 shows the situation at the end of theenrichment process: the plasma port 227 is still open and the piston isclose to the far left position: platelets that have a specific gravitybetween that of plasma and RBCs are trapped at the RBC-plasma boundarylayer 237; the plasma port is about to be closed and the motor stopped.

Typical volumes for the chamber are 20-100 mL, and the amount ofenriched plasma removed at the termination of the procedure isapproximately a quarter to an eighth of the original volume depending onthe degree of enrichment desired.

In order to retain all the platelets and other factors gathering at theRBC-plasma boundary, it is essential to close port 228 before all theRBCs have been removed; otherwise there is the danger of theseconstituents flowing out with the last RBCs. To ensure that this doesnot occur, the blood sample hematocrit value is used to judge theresidual volume of the chamber when the RBC port must be closed. Thisvolume is observable as a piston axial position, and the valve is movedfrom position one to position three as the piston reaches thispredetermined position.

The device described in FIGS. 2 through 6 uses a piston and sealtraveling in a tapered tube, but a right circular cylinder may wellfunction adequately for mixtures of liquids other than blood and wherethe residual volume of the first liquid discharged is not too critical.The tapered tube has the advantages mentioned in the discussion ofFIG. 1. The position of the piston can be judged visually by theoperator relative to graduations on the barrel (not shown), or anoptical detector and automatic valve operation system can be used (notshown).

Since the residual enriched plasma is injected back into the patient thematerials used for this device have to be medical grade materials, atleast for those constituents contacting the blood. Polycarbonate or PTEare suitable for the barrel 206, end cap 215, sleeve 213, frame 208,knob 223 and collar 225. Insert 219 is of a suitable grade of passivatedstainless steel such as 416 or 420. The ball bearings have to do duty athigh speed but operate for very short times so stainless steel bearingsof grade ABMA 1-3 are adequate. O-rings 218 and seal 211 are of siliconerubber. Since the motor does not contact blood, industrial motors (forexample those made by Mabucci) are adequate.

FIG. 7 shows an embodiment with a flexible bladder 312 that initiallyconforms to the bore of the barrel 306, the bladder providing a variablevolume chamber through its ability to invert as shown in FIGS. 10 and11. This embodiment may serve to reduce the effect of entrapped airbubbles.

In FIG. 7 a liquid tight variable volume centrifuge chamber (the BSC) isformed from a tapered barrel 306 containing a molded bladder 312, andend cap 315. The bladder is captured in a return fold 339 between abarrel projection 338 and the end cap 315. Larger end of barrel 306 isclosed by end cap 315. In the case where this device is used to enrichplasma from whole blood, end cap 315 has passages 316 and 317 boredwithin to permit the passage of red blood cells from passage 317 andplasma from passage 316. Passages 317 and 316 connect with valves formedby O-rings 318 compressed in recesses 326 operating in concert withports 328 and 327 respectively in sleeve 313. Sleeve 313 fits slidablyon end cap 315 to permit the ports 328 and 327 to connect with thepassages 316 and 317 at appropriate points in the operation. The knob323 and sleeve 313 can be placed in 3 positions: first position, port328 open and port 327 closed: second position, both ports 327 and 328closed: third position, port 328 closed and port 327 open. Sleeve 313 iskeyed to end cap 315 to permit the transmission of rotary motion betweenthese constituents (key not shown). Insert 319 is fastened to end cap315 to provide an axle for the ball bearing 320 supporting the left handend of the rotating assembly. Since the sleeve 313 is rotating with thechamber a ball bearing 321 is provide to connect the sleeve to anon-revolving knob 323 via collar 325 and rods 322. Barrel 306 isfastened to the shaft 305 of electric motor 301 using screw 307. Noadditional bearings are provided at the motor end, the motor bearingssufficing to support the barrel. The complete assembly is supported by aframe 308, the insert bearing 320 and the motor 301 being located onthis frame. The revolving components all rotate about axis XX. In thisillustration the sleeve is in the first position to keep the port 328open for porting of air as the chamber is charged with blood, and theplasma port 327 is closed. Whole blood 329 fully charges the chamber. Anelastomeric seal 314 permits the introduction of a needle 334 for thepassage of whole blood into the chamber before the start of rotation,and removal of enriched plasma at the cessation of action.

FIG. 8 is a transverse cross section of the device shown in FIG. 7 atsection AA. Whole blood 329 fills the BSC and bladder 312 which is fullyin contact with barrel 306. Frame 308 runs under the rotating assembly.

FIG. 9 shows the device of FIG. 7 in operation rotating at speed. Thesleeve 313 is in position two with both ports 327 and 328 closed. Theboundary between RBCs 331 and plasma 330 is shown at 337. The bladder isstill against the barrel now under the influence of the pressuredeveloped by the spinning liquid mixture.

FIG. 10 depicts the situation after spinning for 60 seconds or so. Thesleeve 313 is placed in position one, port 328 is open and RBCs 331 arebeing discharged through port 328. Plasma port 327 is closed. Thebladder has moved to the left to compensate for the volume of RBCs thathave been discharged. The shape adopted by the bladder is a balancebetween the forces developed by liquid pressure pushing the bladder tothe right and atmospheric pressure (via vent 332) pushing the bladder tothe left. Since the pressure at the center of the spinning liquid isnear absolute zero the atmospheric pressure exceeds the left handpressure that has been developed up to a certain radius, hence there-entrant shape of the bladder. The volume of plasma 330 has remainedthe same as when introduced. The boundary between RBCs and plasma isshown at 337. In this view the RBC discharge is about to be stoppedsince the residual RBC volume 331 is low enough.

FIG. 11 illustrates the final position for the bladder 312 while therotation continues but just prior to stopping. Sleeve 313 is in positionthree, RBC port 328 is closed and plasma port 327 is still open. Plasmahas been discharged through port 327 and is about to be cut off by thebladder rolling onto end cap 315 and cutting off the passage 316. Thisillustrates the minimum volume of enriched plasma 330. At this point thesleeve 313 is moved to position two with both ports closed and therotation is then stopped; the residual liquid is removed using a syringein a similar manner to the charging described in FIG. 7.

Materials for the device of FIGS. 7 through 11 are similar to those forthe device of FIGS. 2 through 6: the bladder by example can be made ofsilicone rubber, polyurethane or polyvinylchloride.

For the previous device 200 the piston position provided the signal forclosure of the RBC port 328. In the case of the bladder the invertedbladder rolls along the tapered barrel bore, the axial position of thereverse edge providing (labeled 312 in FIG. 11) the volume and thesignal for port closure. The cut-off of the plasma discharge isautomatic as the bladder rolls over the port passage 316.

The device described in FIGS. 12 through 16 utilizes an air core anduses no bladder or piston.

The device of FIG. 12 is very similar in construction to the twoprevious embodiments, with a BSC formed from a barrel 406 and end cap415. The inner surface of the end cap 415 forms the larger diameter endwall of the chamber, with the inner surface of the barrel 406 formingthe chamber's tapering side wall. In this illustration whole blood 429from syringe 433 fills the centrifuge chamber through needle 434 withboth ports 428 and 427 closed. Air displaced by the blood leaks outthrough the clearance between the needle 434 and insert 419 bore as theblood is injected. FIG. 13 shows the circular section nature of FIG. 12.Once the charging syringe is removed, the motor is started and thechamber is rotated at 10,000 to 20,000 rpm for approximately one minute.At this point the sleeve 413 is moved to the second position, and RBCsare discharged through port 428 until the point shown in FIG. 14 wherethe minimum RBCs 431 remain. Meanwhile, the plasma adopts the region orlayer 430, and a boundary 440 forms at the plasma-air radial interface,the air core 438 having entered through the bore of insert 419 (via afilter in the housing not shown, but see FIGS. 17 and 18). At thisjuncture the sleeve is moved to the third position, port 428 closed andport 427 opened. With this preferred device there is no bladder orpiston to observe, so the operator observes the axial interface 436between the RBCs 431 and the plasma 430 of the mixture through thetransparent barrel to determine when to manually close the RBC port 428and open the plasma port 427. With blood, this mixture interface is easyto see and can be automated with an optical detector. The difference inelectrical resistivity between red blood cells and plasma can also beused to trigger an indicator or automated valve. An alternative way ofdetermining the point at which to shut the RBC port is to use time.After one minute of running to separate the constituents of the blood,the RBC port is opened and a timer started. Since the pressure generatedin the centrifuge is a predictable function of liquid specific gravityand running speed, and since the RBC port is a precisely calibratedorifice, the flow rate being discharged, and hence time can be computedfor a given hematocrit value.

With the motor still running, the plasma discharges through port 427until it reaches the situation in FIG. 15 where the residual RBCs are atlayer 431 and the residual plasma at layer 430. The sleeve is then movedto the second position to close both ports. In the case of plasma thepassage 416 is placed at a precise radial location to give an accuratefinal volume since no further flow of plasma will occur once the aircore 438 has grown to that passage radial location. The motor is thenstopped and the device placed on end, with the motor downward, so thatthe rotation axis is vertical as shown in FIG. 16. The remainingenriched plasma with some RBCs is removed by syringe and needle asillustrated.

An enclosure suitable for various embodiments discussed in thisapplication is described in FIGS. 17 and 18; however these two figuresshow the enclosure applied specifically to the air core embodiment ofFIGS. 12 through 16. The frame 508 is mounted to a battery power pack503 that acts as the base for the enclosure. An outer casing 500surrounds the centrifuge and is fastened to the battery pack 503, thejoint being liquid and air-tight. A valve selector knob 545, integralwith eccentric 546 and pin 547, is mounted in the casing such that theselector knob 545 can be turned by the operator to actuate the internalknob 523 via the pin 547 in groove 548 and hence the collar 525 andvalve sleeve 513. In FIG. 17 the motor 501 driving the chamber BSC iscontrolled manually by switch 504 connected to battery pack 503 by wires550. A bush 543 mounted at the left hand end of the enclosure 500provides alignment for the entry of the syringe (433 of FIG. 12) needlewhen charging the chamber with whole blood or when extracting theenriched plasma. Immediately adjacent to bush 543 is a porous flexiblepierceable filter 544. This filter has two functions: It filters the airentering the core of the centrifuge when it is running, and it preventsthe egress of any aerosols into the atmosphere of blood fragmentsgenerated as the centrifuge discharges RBCs or plasma into the casing. Asmall slit in the filter allows the charging syringe needle to enterwithout damaging the effectiveness of the filter. Covering most of theinterior walls of the casing 500 is a highly absorbent lining 542 toabsorb the RBCS and plasma discharged into the casing as the air core538 enlarges and the enrichment process proceeds. A lens and mask 549placed in the wall of the casing 500 permits the operator to view theaxial interface 536 of the RBCs and plasma as the process of enrichmentproceeds. The mask and lens are chosen to enhance the contrast of theimage seen of the liquid separation interface 536.

A photo detector (not shown) can be placed in the location of the lensto provide an electrical signal of the progress of the liquid separationinterfaces, and an electromagnet actuator can drive the valve selectorknob 545. These electrical elements in conjunction with a manual switchcan be used to control the entire process once the motor has started.

From tests to date it would seem feasible in some applications to use asimple timer program to schedule the sleeve motions. For example, thefollowing sequence can operate off a timer once the chamber is chargedwith blood, a) start motor, run for 60 seconds b) open RBC port anddischarge RBCs for 30 seconds, c) close RBC port and open plasma portand run for 30 seconds, d) close both ports, and stop motor. Such adevice might require the addition of a means of manually inserting thepatient's hematocrit number to allow for varying proportions of RBCs toplasma.

Table 1 gives typical data obtained for the air core device of FIGS. 12through 16 using porcine blood. The data was obtained with runs of oneminute for the initial separation and approximately one more minute todischarge the RBCs and plasma.

TABLE 1 Platelet % Red Platelet Count Concentration % Platelet BloodCells Sample (×10³/microliter) Factor Recovery Removed Baseline 229 NANA NA Run 1 1656 7.2 100 93 Run 2 1457 6.4 88 92 Run 3 1446 6.3 87 93Run 4 1685 7.3 100 94

For all three embodiments discussed, piston, bladder and air core, thesize and position of the ports and passages are very important. As thecentrifuge rotates, the pressure developed within the chamber varies asthe square of the speed and the square of the radius of rotation. Togain manual control over the discharge of constituents the dischargeneeds to take place over a manageable time. The RBC port for exampleneeds to be sized to allow passage of the RBCs over a period of about 30seconds. Conditions must be selected to allow the RBC port to functionwithout blockage as the RBCs try to clump, and flow has to be kept lowenough to stop the platelets from being swirled into the exit vortex.For centrifuges using whole blood samples of approximately 30 mL, it hasbeen found that RBC ports of the order 0.008 inch diameter work well ifspeeds are in the region 15,000 to 20,000 rpm and chamber barrels areabout 1.0 to 1.25 inch in diameter at the largest point. Plasma portscan be larger since the risk of losing the platelets is less: values ofabout 0.010 inch diameter are adequate. Placement of the plasma portsrelative to the center axis of rotation has a direct effect on theattainable concentration factor. The closer to the center, the lessplasma is removed and less concentration is achievable. Additionally, invarious embodiments of the invention discussed it will be noticed that asmall annulus 241, 341, 441, 541 is created at the large diameter end ofthe chamber. This annulus creates a localized area of increased radialdepth, but of small volume, for the RBCs prior to their entry into theRBC passages 217, 317, 417. This increase in depth reduces the tendencyfor the platelets and other desired factors from exiting with the RBCsbeing discharged through the RBC port 228, 328, 428 under influence ofthe exit vortex created locally close to the same ports (not shown).

In all the embodiments discussed the accuracy of the RBC port closurepoint can be improved by employing a separation aid, such as a flowableseparator gel of an intermediate specific gravity between the red bloodcells and the platelets. The separator gel spreads over the red bloodcell layer moving the other layers further towards the center axis. Theseparator gel automatically caps the first port when all of the redblood cells have exited. The separator gel viscosity is designed so thatit will not pass through the small exit port at the centrifuge speedemployed in the BSC. The automatic shut off of the first port can alsobe accomplished with a separation aid in the form of a solid material ofintermediate specific gravity that is designed to enter and close offthe port when the red blood cells have fully exited. An example would beplastic beads such as microspheres with the desired intermediatespecific gravity that are large enough to cap the port when agglomeratedas they flow toward the port.

For the bladder and air core embodiments the visualization of the RBCplasma axial boundaries can be improved by incorporating back lighting,such as in the form of an LED mounted inside the BSV adjacent to themotor centerline. Additional windings in the motor could provide the lowpower needed to power the lamp.

With adjustments to size and locations of the port and passagedimensions, the subject invention also has the capability for separatingand concentrating a wide variety of therapeutically beneficial cells andother biological constituents. Many of these biological constituentshave the potential for regenerative therapy and can be characterized asregenerative agents. These regenerative agents can assist with theregeneration, restoration, or repair of a structure or assist with thefunction of an organ, tissue or physiologic unit or system to provide atherapeutic benefit to a living being. Examples of regenerative agentsinclude for example: stem cells, fat cells, progenitor cells, bonemarrow, synovial fluid, blood, endothelial cells, macrophages,fibroblasts, pericytes, smooth muscle cells, uni-potent and multi-potentprogenitor and precursor cells, lymphocytes, etc. The invention also hasthe potential to process soft or liquid tissues or tissue components ortissue mixtures including but not limited to adipose tissue, skin,muscle, etc. to provide a therapeutic regenerative agent. The resultingseparated or concentrated products from the various embodimentsdescribed herein may be used as is known in the art. Medical treatmentprocedures may call for the concentrated product to be applied directlyto a treatment site, or incorporated into a treatment device (e.g.,administered to an absorbent implant material prior to, concurrent with,or post-implantation), or even combined with another material as amethod of treatment, for example, by combining with a particulatematerial to form a paste (e.g., combined with a extracellular matrixthat has been formulated as a powder).

The blood centrifuge container may also incorporate an adjustable port,e.g. a tube with an open end extending radially into the BSC and hingedat the outer periphery in such a manner that the tube can be swung in anarc for the open end to scan a range of radii (not shown). The locationof the open end of the tube can be adjusted before or during operationsuch that it is located at a desired position with respect to the axisof rotation. For example, the entrance port could be located towards theperiphery of the centrifuge container to initially vent undesired cells,and later adjusted towards the center of the container to vent plateletpoor plasma. Alternatively, if the plasma fraction is what is desired tobe removed, the port can be positioned so that essentially only plasmais tapped from the stratified mixture.

The apparatus may also be configured to shut off, or at least to ceaserotating, once a predetermined quantity of one or more constituents suchas plasma has been tapped. Specifically, a port may be positioned suchthat, upon stratification, the plasma constituent is adjacent the port.When the valve for that port is opened, plasma is dispatched out throughthe port. The port may also be configured with a sensor that senses thepresence or absence of plasma. As such, the apparatus can be configuredsuch that the barrel continues to rotate as long as plasma is sensed ator in the port, but when plasma is no longer sensed, the sensor providesa signal to the motor to stop (thereby stopping the rotation of thebarrel) or signaling the opening of a tap. As plasma continues to beremoved from the barrel through the port, eventually the supply ofplasma at the radius of the port is exhausted, thereby causing a signalto be sent from said sensor, and the barrel stops rotating. Of course,each of these signals may arise from the sensing of any stratifiedlayer, not just plasma.

It may be desirable to collect one or more of the discarded fractions ofthe liquid specimen in addition to the concentrated fraction. This canbe accomplished by one of several methods. A collection bag or chambercan be connected to an exit port on the sleeve. This bag or chamber willrotate with the barrel so provisions must be taken to balance it aroundthe axis of rotation. Another method would be to have a circumferentialfunnel opposite the desired exit port that would collect the fractionbeing discharged and guide the fluid to a collection point by gravityflow. This is further illustrated later in reference to FIG. 25.

Further embodiments are shown in FIGS. 19 through 26. These figuresdescribe a device using the air core principle covered in FIGS. 12through 17 but incorporating improvements designed to maximize theenrichment obtainable when preparing PRP. FIG. 19 shows the two majorcomponents of a centrifuge designed to be used in two components, areusable drive unit 601 and a disposable portion 600. The separation ofthe centrifuge into two components allows the disposable component to bemore cost effective.

FIG. 20a is a schematic representing a half mirror section of arevolving chamber defined by the boundary letters ‘defg’. Significantdimensions are noted by length references L1 through L8, and the radiiidentified as D1 through D8. As can be seen in FIG. 20a , D1 correspondsto the length of the radius measured from the rotational axis XX to theouter end of the channel 640, as shown in this embodiment having anoptional plenum at the end of the channel, where exiting RBCs 641 enterinto the RBC passage 639. Similarly, D2 and D3 identify the innerdiameters for the right and left sides, respectively, of the plenum atthe end of the channel 640 farthest from axis XX. D4 and D7 mark theouter and inner diameters, respectively, of the flat located on theright hand end of wedge 609. D5 identifies the diameter at the interfacebetween the red blood cells 641 and the buffy coat 642. D6 identifiesthe diameter at the interface between the buffy coat 642 and the plasma643. D8 identifies the inner diameter of plasma passage 610, andcorresponds to the interface of the plasma 643 interface with the aircore 646. The length measurements L1 through L8 are based upon adistance measured from the reference line corresponding to the rightside of the plasma passage 610. L1 and L2 are measured to the left andright hand sides, respectively, of the plenum at the end of the channel640. L3 identifies the length to the flat on the right hand side of awedge 609 (to be described later), measured from the reference line. L4and L5 identify the location of left and right markers 644. L6corresponds to the length to the edge of the rotation chamber measuredat the diameter corresponding to the buffy coat/plasma interface D6. L7corresponds to the length to the edge of the rotation chamber measuredat the diameter corresponding to the inner diameter of the flat locatedon the right hand edge of wedge 609. L8 corresponds to the length to theedge of the rotation chamber measured at the diameter corresponding tothe inner diameter of the entry into the plasma passage 610.

The rotational axis XX passes through boundary ‘dg’. The major crosshatched area represents the tapered chamber with the outer wall having ahalf angle ‘a’. Inserted into the conical recess of the chamber is thewedge 609 having an external frusto-conical portion of half angle ‘b’that defines RBC channel 640 and an internal reverse frusto-conicalrecess defining half angle ‘c’ that defines the boundary of the plasma643. It should be noted that half angle ‘b’ need not necessarily be thesame as half angle ‘a’, in other words the channel 640 may be tapered,not parallel.

As fluid exits the RBC outlet port, the fluid exiting through the RBCpassage 639 experiences high shear forces, and the RBC channel 640serves to ensure that the RBC passage 639 entry port is at the end ofthe channel 640 and at a distance removed from the RBC-BC interface,with the channel dimensioned to allow for significantly slower localflow speeds at the RBC's entrance into the channel 640, relative to thehigh exit speed the RBC experiences as it exits through the RBC passage639.

For example, in one embodiment, RBCs collect at the outer edge of thespinning chamber and discharge through one or more RBC passages 639 fedfrom a circumferential groove or plenum, which, in turn, is fed from athin circumferential channel 640, or alternatively, circumferentialsections forming multiple channels 640, starting adjacent to thebuffy-coat collection areas. The circumferential channel 640 has acircumference many times larger than the radial depth of the channel.For a device providing a 60 Ml centrifuge, and having a channel with a4.5 inch circumference by 0.020 radial depth the orifice diameter forRBC passage 639 would be of the order 0.010 inch. This combinationspinning at approximately 17000 RPM would result in velocities of2000-3000 cm/sec from the orifice at RBC passage 639, and only 1.5cm/sec along the channel 640. Thus the channel 640 slows the flowadjacent the separation layer by a factor of over 1000 to 1. In anotherembodiment (not shown) not having a plenum, the RBC passages may be feddirectly from the thin circumferential channel, starting adjacent to thebuffy-coat collection area. Similar performance, in achieving areduction of flow rate at the separation layer, when compared to theorifice exit, would be expected as that described with reference to theembodiment having a plenum.

It has been observed that there may be a benefit in evacuating the RBCsunder a reduced rotational speed of the spinning chamber. This reductionof rotational speed must be accomplished in a manner that does notdisrupt the stratification of the separated constituents, further; thereduced rotational speed must not be reduced to the point of allowingsignificant degradation of the established stratification of theconstituents. For example, upon achieving satisfactory stratificationthrough the operation of the device at a first speed suitable forseparation, a gradual ramping down of the rotation speed will maintainthe stratification, and once arriving at a second rotational speed, theRBC cells may then be ejected through the RBC passage 639, at acorrespondingly reduced velocity as a consequence of the lower forcescreated through the reduced rotational speed of the spinning chamber.For the example previously described, having a rotational speed ofapproximately 17000 RPM for separation, the gradual reduction may occurin a controlled fashion over a determined period of time, until settlingat a targeted lower rate of rotation, in this new example rotating atapproximately 13000 RPM, in order to allow evacuation of the RBCs whilestill preserving the integrity of the RBC/BC interface. It is alsorecognized that minor adjustments to the timing of these steps may, forpractical purposes, may achieve similar results, such as opening of theRBC valving while the speed is still ramping down, but close to thetargeted evacuation rate.

Modifications to the dimensions, or rotational speeds may be employed toensure that a reduction in localized flow rates, when measured at theRBC passage 639 and compared to the RBC entry into the channel 640, maybe made to achieve different reduction rates, such as reduced beyondapproximately 500:1, or 100:1, instead of the 1000:1 described above. Ascan be seen in the embodiment of FIG. 20a , the channel 640 is arrangedon a radially shallow angle a, and is shown having a plenum at theterminus of the channel, from which the RBC passage 639 provides for thedischarge of the RBC. In another embodiment (not shown), the device maynot provide a plenum at the terminus of the channel, but rather thechannel terminus may include the outlet for the RBC passage, or thechannel may reduce in dimension (taper) and funnel directly into theoutlet for the RBC passage. As described above, the devices of thisinvention aim to reduce the effect of the exiting RBCs upon the buffycoat components, as may be accomplished by providing for spatialseparation between the RBC outlet and the RBC/buffy coat interface. Itis this spatial separation, with or without a plenum in the channel,that reduces the tendency for the platelets and other desired factorsfrom exiting with the RBCs being discharged through the RBC passage 639under influence of the exit vortex created locally close to the port. Byoperating the device in a manner that prevents plasma or buffy coatcomponents from entering the channel 640, the high shear forces will belimited in effect only to the RBC component, and will be unable todisrupt the interface between the RBC and the BC. Typically, whencomparing the concentrated blood product with the starting material,about 93% of the RBC's are removed using the chamber as described above,and as depicted in FIG. 20 a. It may be desirable in some instances toremove an even greater proportion of the RBC's. Values of approximately98% removal have been achieved by further managing the turbulent flowtowards and directly above RBC passage 639. The management of theturbulent flow above and adjacent to the RBC passage may be achieved insome embodiments by, at least partially restricting or even completelyclosing off the flow into the circumferential channel (in a direction offlow that is generally from the flat of the wedge, towards the plenum,if any), without preventing the flow of fluid through the channel (in adirection that is largely perpendicular to, and circumferential around,the axis of rotation) towards the RBC passage. This restriction may becreated over some portion of the circle that is the circumferentialchannel. For example, in an embodiment, the width of channel 640, atleast the portion closest to the RBC passage, can be at least partiallyreduced, for example, in one embodiment, from at least 1% to 100%, inanother embodiment from at least 10% to 100%, or in yet anotherembodiment, from at least 20% to 100%, and in still another embodiment,from 50% to 100% reduction, over a portion of the circumferentialchannel, for example, in an embodiment from about 10 degrees to about350 degrees, or, in another embodiment from about 15 degrees to about270 degrees, or, in still another embodiment, from about 20 to 180degrees of the circumference, with the angular center optionally beingsubstantially aligned with the location of the RBC passage 639. FIG. 33depicts a restrictive feature 800 that restricts the flow of fluid intothe channel 640, and as shown in FIG. 33a , is in the portion of thechannel encompassed by angle d (here depicted as 90 degrees) of thecircumference. The restrictive feature may be integrated as part of therotating chamber, such as by machining or molding of the rotationchamber, or alternatively may be manufactured as a separate componentand later affixed in some manner known to those skilled in the art, toeither the wedge 609 surface (as shown in FIG. 33), or alternatively, tothe interior surface of the rotating chamber (not shown). Therestriction may be of uniform dimensions, as shown in FIG. 33a , oralternatively, in an embodiment (not shown) there may be provided avariable reduction in the channel 640, where the greatest restriction ofthe entrance into the channel is at the portion of the channel that isaligned with the RBC passage, and the restriction percentage is reducedin a gradual or steep taper, or even a stepped manner, as the distanceof the channel increases from the RBC passage. For this embodiment, thegoal is to provide the appropriate percentage restriction tailored tocounteract the variability of the flow rate into the channel arisingfrom the variable proximity to the RBC passage; thus in the regions ofthe channel closest to the RBC passage, the percentage of restriction tothe channel will be maximized, while away from the RBC passage, thepercentage of the restriction of the channel will be appropriatelyreduced, thus by ensuring uniform flow rates into the channel, thedisturbance to the interface between the separated layers (e.g., RBC/BCinterface) is minimized. In these, and any other embodiment of thecentrifuge devices described herein, it is recognized that by usingmaterials in the construction of this part of the chamber that aresimilar in density to blood, a condition of imbalance for the rotatingchamber is avoided, or alternatively the rotating chamber may bebalanced using counter weights, properly placed, as known to thoseskilled in the art.

Similarly, by placing the plasma passage 610 at a location removed fromthe buffy coat component (and optionally located within a plenum asdepicted in FIG. 27a ), and with the buffy coat-plasma interface notextending inward beyond D7, the buffy coat can be contained within thechamber, as with the shallow angle c, the high shear forces at theplasma passage 610 will not cause the disruption of the BC-plasmainterface. Thus there is a reduction in the tendency for the plateletsand other desired factors from exiting with the plasma dischargedthrough the plasma passage 610 under influence of the exit vortexcreated locally close to the port. Though depicted in FIG. 20a aslocated at the base of the wedge 609, the plasma passage may be locatedelsewhere, so long as the opening is at a suitable radius that issmaller than the radius of the buffy coat-plasma interface, such as at alocation corresponding to L8 in FIG. 20a . Through these features, theembodiments described aid in preserving substantially all of the buffycoat component within the chamber and enhancing concentration orenrichment efficiency of the finished product.

Furthermore, with reference to FIG. 27a , there is depicted anembodiment identical to that shown in FIG. 20a , except that there isincluded a plasma plenum 655 in the form of circumferential groove (orportions of a circumferential groove) housing the orifice(s) that leadinto the plasma passage 610. In this embodiment, the exiting plasma willflow along the tapered channel defined by the boundaries of the wedge609, and the air core interface with the plasma. While the chamber isbeing rotated, and the plasma valve open, the plasma will flow towardsthe plasma passages (depicted here located at the base of the wedge609), and spill over the wedge base and into a plasma plenum 655. Oncewithin the plasma plenum, the plasma will flow along the length of theplenum (i.e. circumferentially) until it encounters and exits throughthe orifice(s) leading to the plasma passage 610. While the plasma istraveling within the plenum 655, it will not exert shear forces upon theplasma/buffy coat interface, which is at a distance removed, andphysically shielded by the presence of the wedge 609.

Comparing the FIGS. 20b and 27b will allow visualization of thedirection of fluid flow as the plasma approaches the plasma outlet,whether as a continuous slope (the geometry shown in FIG. 20b ), or witha plenum 655 (the geometry shown in FIG. 27b ). These figures representa projection view, looking down towards the opening to the plasmapassage 610, as if one is looking from the axis of rotation towards theoutside diameter of the chamber.

With reference to FIG. 20b , the plasma is depicted as traveling fromright to left, and as the fluid approaches the left edge of the chamber,the fluid will be drawn towards the outlets for plasma passage 610. Inthis embodiment not having a plasma plenum, the shear forces will beproportionally reduced with increasing distance from the opening, thusas the plasma travels along the inside face of the wedge (along anglec), the shear forces will not necessarily be uniform throughout theentire diameter of the region, but will be higher when alongside thelocations of the openings to the plasma passage 610. While the geometryof FIG. 20a has been empirically determined to be effective inminimizing shear forces affecting the buffy coat/plasma interface, itmay be possible to even further reduce the shear forces experienced atthe flat of the wedge during the operation of the device.

With reference to FIG. 27b , the plasma is depicted as traveling fromright to left, and enters into the plasma plenum 655, prior to flowingalong the plenum towards the openings 610. As can be seen by the uniformarrows (right side) depicting fluid flow towards the plenum 655, thepresence of the plenum is expected to reduce variations in shear force,when measured circumferentially within the plasma channel (the plasmaflowing between the wedge face at angle c and the air core), as theplasma will approach the base of the wedge 609, and flow into the plasmaplenum 655, and thus create an effect similar to water flowing over thebreast of a dam. That is, prior to cresting the obstruction, whetherupstream of the dam, or prior to entering the plenum, the fluids flowslowly and smoothly, then once past the obstruction, whether downstreamof the dam or within the plenum, the fluid flow rates will be relativelymuch higher and less uniform. As can be seen by the arrows depicting thefluid flow pattern, the flow of plasma towards the plasma plenum isexpected to be uniformly distributed over the entire diameter, then oncethe plasma has crested the wedge, and is within the plenum 655, thenthere will be large variations in fluid movement as the plasma flows outthe one or more openings to the plasma passage 610. Since the variabledirection shear forces are largely contained within the plenum, and notaffecting plasma flowing along the wedge face, this embodiment would beexpected to allow for enhanced enrichment factors of the buffy coatcomponents. The geometry of this embodiment allows for retained plasma,measured as the depth between D8 and D6, to be minimized, due to thereduced variability of plasma flow rates, when measuredcircumferentially along the plasma channel, which would otherwise tendto disrupt the buffy coat/plasma interface.

Furthermore, with reference to FIG. 20a , it should be pointed out thatthe volume of plasma remaining after all the discharged plasma has leftthe chamber is defined by the boundary diameters D8 and D6. This volumecan be tuned to get the value of enrichment desired by adjusting thesesame mentioned dimensions.

It should also be made clear that to obtain high degrees of enrichment,the depth of plasma beneath the buffy-coat (as seen in FIG. 20a ) mustdecrease (diameter dimension (D6−D8)/2 decreases) so the risk ofplatelet loss increases because the out-flowing plasma shears thebuffy/plasma interface more closely. However, the pressure driving theplasma outflow gradually drops to zero as the plasma diameter approachesD8 since pressure driving the plasma flow is proportional to the squareof the speed of rotation, multiplied by the difference of the squares ofthe radius of the opening of the plasma passage located at D8 and theradius of the plasma/air interface within the chamber.

By taking advantage of this steadily reducing flow effect as the plasmaapproaches D8, the plasma depth (D8-D6) can be minimized, with littleloss of buffy coat due to shear, and the residual plasma volumeminimized and the enrichment maximized.

To summarize, RBC/buffy-coat shear is minimized using the outer diameterchannel to control RBC/buffy-coat shear, and plasma/buffy coat shear iscontrolled by geometry and the reducing plasma to air core drivingpressure.

Thus, while the chamber is rotating, and prior to the discharge of anyof the plasma, there is a larger pressure head driving the plasma outthrough the plasma outlet and into plasma passage 610, subsequently, asthe volume of plasma in the chamber decreases, the pressure head abovethe plasma outlet is reduced in a proportionate amount, until the plasmalevel reaches the level of the plasma outlet at D8, and all plasma flowout through the plasma passage 610 terminates. As the flow rate throughthe plasma passage 610 is reduced as the plasma volume is reduced, thisprovides the added benefit that the tendency for shear forces to affectthe buffy coat is minimized, as at the point the plasma flowing out andthe buffy coat are at nearest proximity to each other (i.e., thedistance between D6 and D8 is at its minimum), the plasma evacuationflow rate will be at its lowest rate.

In operation blood fills the chamber and after a period of time at speedseparates in to red blood cells (RBC), buffy coat and plasma. Afterseparation, RBC passage 639 is opened and RBCs discharge from RBCpassage 639, the interface of the RBC's being evident at L5 at thetransparent conical surface. Visible markers are placed on the chamberat L5 and L4 to guide an operator in the closing of RBC passage 639:when the RBC interface reaches somewhere between L5 and L4 the dischargeof RBC's out of RBC passage 639 is stopped by manipulation of valves tobe described later. At this point, residual RBCs occupy a predefinedvolume defined by the conical channel 640 and the circumferential recessat the left hand end of the RBC channel 639. When collecting buffy coat(BC) 642, defined on the illustration by the honeycomb hatch, it isimportant to prevent the BC from migrating into the RBC channel 640,since the BC cannot be recovered at the end of the procedure if theymigrate there. To ensure that this does not happen, the rate at whichthe RBC interface appears to move along the conical surface of thechamber is controlled to a velocity that is sufficiently low for anoperator to stop the process (by closing RBC passage 639) as theinterface travels between makers placed at L5 and L4. This velocity is afunction of speed of rotation, diameter of the chamber, size of the RBCdischarge port connected to passage 639, and the half angle ‘a’ of thechamber. These variables are adjusted to give an interface velocity atL5 or L4 that is manageable by a human operator but that does not impedethe rapid separation required (whole process of separation, discharge ofunwanted RBCs and plasma in less than 2 minutes). In testing variousparameters, it has been experimentally determined that an interfacevelocity of approximately 4 mm/sec allows accurate intervention by theoperator, though it is recognized that higher and lower velocities maybe desirable, on the range of less than 10 mm/sec. (In the case wherethe RBC to Buffy coat interface is detected by optical sensors or thelike the approach velocity of the interface can exceed the 10 mm/sec.rate). When RBC port 638 is initially opened, there is a potential fortemporary turbulence due to the sudden pressure drop that may cause somedisruption of the clarity of the interface between the RBC and BC, atD6. The effect of this turbulence can be minimized by an automatic rampdown in the centrifuge speed that is controlled by the software in thebase unit 601. Changes in centrifugation speed can be programmed in thesoftware to automatically initiate by timers in the software or signalsgenerated by movement of the valve mechanisms. When the RBC discharge isstopped, the BC is captured at the end of the flat or separation surfaceon the right hand end of the wedge 609, defined by diameters D4 and D7.Though the separation surface is depicted in FIG. 20a as being at 90degrees to the axis of rotation, it is envisioned that the separationsurface may be at another angle relative to the axis of rotation. Theseparation surface forms the “top” surface of the wedge 609 when thecentrifuge is in its normal upright orientation. If the RBCs are stoppedat L5, the BC outer diameter is D5, if the RBCs are stopped at L4 the BCouter diameter is at D4. The buffy coat (BC) volume is around 0.5% ofthe blood volume initially introduced, so the flat on the end of thewedge (D4, D7) can be defined to ensure that in the worst case (RBCstopped at L5) the BC stays on the separation surface and does notextend into the inner half angle cone ‘c’. Once the RBC passage 639 isclosed, the plasma passage 610 is opened and plasma flows to discharge.The illustration shows the situation when all the plasma has flowed outof plasma passage 610 and flow has stopped because the air core 646 hasexpanded to the diameter of the passage inlet at D8. Prevention of BCgetting into the inner cone is important since the axial velocity of theplasma surface accelerates as it approaches the exit passage 610 andfast shear velocity at the BC/Plasma interface results in loss ofplatelets into the plasma. With radial separation of BC to air core(D6−D8)/2 of the order 1 mm-2 mm, the loss of platelets into plasma isacceptable and enrichment factors (EF) of 8:1 or more can beconsistently obtained. Enrichment factors are defined by the followingequation: (EF=(# of platelets captured in the BC sample per unitvolume)/(# of platelets in the original whole blood sample per unitvolume)). Fundamentally, this design has been conceived to minimize theshear at the RBC/BC and BC/Plasma interfaces and hence reduce loss of BCto the RBC discharge or the plasma discharge.

In one embodiment, the orientation of the device in use is with the axisof rotation XX being vertical, with the port valve 602 at the top of thedevice. As a consequence of the geometry of the rotating chamber, whenthe rotation is halted, any fluid (e.g., RBC) that is within the channel640, will tend to remain contained in that channel, and substantiallyall other fluid above the line corresponding to the flat 608 of thewedge 609 while in operation (i.e., to the right of L3 in FIG. 20a ),will flow by gravity, upon cessation of rotation, and pool directlyunderneath the port valve 602, and is available to be harvested, such asby being drawn into a needle directed through the port valve and intothe pool of concentrated materials. It is recognized that the variousembodiments described herein may be operated at another angle (e.g.,horizontal), and then optionally rotated to vertical for harvesting,after cessation of rotation. By maintaining the RBCs sequestered withinthe channel 640 upon cessation of rotation of the chamber, theconcentration of the buffy coat components can be maximized, as thosematerials within the channel (e.g., RBCs) are not available to furtherdilute the concentrated buffy coat or other blood components. In someembodiments, it may be advantageous to add a surface tension modifyingcoating (e.g., hydrophilic or hydrophobic coating) to at least a portionof the rotating chamber, such as the flat 608 at the end of the wedge609 to prevent some of the captured BC from remaining on the flat due tosurface tension. Furthermore, there may be a benefit in providing anangle (e.g., 1 to 45 degrees) to the flat of the wedge, in order todirect the flow of fluid towards the central collection area, if in agenerally vertical orientation.

It has been observed that causing the rotation chamber to deceleraterapidly, or alternatively abruptly, or with an uneven rate of decrease,will lead to an increase in the amount of platelets in the collectionarea, relative to a more gradual deceleration of the rotation chamber.It is believed the rapid deceleration, such as may be accomplished byincorporating a braking system into the device, will create mixing ofthe components above the flat of the wedge, and avoids the occurrence ofresidual concentrated buffy coat components remaining on the surface ofthe flat of the wedge. It is believed that the red blood cells remainingwithin the chamber and within the channel, will remain largely containedwithin the channel, and not mix with the buffy coat, even upon rapiddeceleration. Alternatively, one may simply physically dislodgeplatelets, such as by tapping, shaking, jarring, or otherwise disturbingany components that, due to surface tension, had remained away from thecollection area, such that they can now be collected.

The geometry of the embodiments of the device incorporating the wedge609 provides at least 3 benefits aiding in the efficiency, and operationof the device, as the wedge 609 serves to: 1) create spatial separation;2) form the channel; and 3) increase the apparent depth of liquids.First, the wedge creates spatial separation between the outlets for theplasma and the RBC, and therefore can minimize the effects of shearforces at the outlet from affecting the buffy coat components whichremain distant alongside the flat of the wedge. Second, the wedgepartially forms the channel, as the outermost surface of the wedge, atangle b, provides part of the inner boundary of the channel 640. Third,the wedge enhances the ease of operating the device, as it enhances theapparent depth of the liquids displaced by the existence of the wedge.That is, the wedge serves to displace the volume of the fluids that arein the wedge region (between D2 and D8), and has the effect ofincreasing the apparent depth of these liquids, as dimensions between D4and D5 are increased due to the displacement, and necessarily thespacing between markers 644, at L4 and L5, can accordingly be madelarger and provide greater resolution for the operator. With the effectthat the operator can now more accurately determine when to halt thedischarge of the RBC through the RBC passage 639.

FIG. 21 shows the device of FIG. 19 assembled and in the running statewith the RBC port 638 open, the plasma port 612 closed, and the RBCdischarged to the RBC-Plasma receiver 647. No plasma 643 has yet beendischarged to the receiving chamber. The air core 646 is fullyestablished and the separated fluid components are established withclear boundaries. The spinning centrifuge blood-containing chamber ismade up from two elements, the tapered barrel 606 and the end cap 614.This chamber spins in two bearings 619 and 604, the smaller bearing 604locating the narrow end of the chamber, and the larger bearing 619locating the larger end of the chamber indirectly via the drive shaft617 and the valve cap 616. The smaller bearing 604 is mounted in thetransparent mid cover 607 and the larger bearing in follower 618. Valvecap 616 rotates with the chamber components driven by a key or pin (notshown) from the end cap 614, and can translate axially along therotation axis propelled axially by the follower 618, which in turn ismoved axially by cam followers 620 and cams 621. Axial movement of valvecap 616 controls the position of RBC port 638 and plasma port 612, andthus controls the discharge of RBCs from RBC passage 639 or plasma fromplasma passage 610. Cams 621 (typically 3 in number but may be more orless than 3) are integral with drum 613. Follower 618 can move axiallywithin drum 613 but is prevented from rotation by male keys 631 on thefollower and female keys on substructure 624. By rotating drum 613 theoperator moves follower 620 axially and thus controls the position ofthe RBC and plasma ports 638 and 612. RBC-plasma receiver 647 surroundsthe rotating elements to capture the discharged RBCs and excess plasmaand moves axially with the valve cap 616.

Clearance between shaft 617 and valve cap 616, and the clearance betweenvalve cap 616 and end cap 614 affects the fit and concentricity betweenend cap 614 and valve cap 616. ‘O’-rings 648 and 611 act as seals and/oract as suspensions between these two caps. If the clearances are heldvery small the ‘O’-rings act only as seals, but if the clearance isincreased substantially the ‘O’-rings do double duty as seals and assuspensions. Such suspension characteristics can be selected so that thenatural frequency of the valve cap 616 oscillating on the chamberassembly (shaft 617, end cap 614, and barrel 606) is substantially loweror substantially higher than the operating speed.

Centrifuge coupling 633 attached to drive shaft 617 accepts torsionaldrive from motor 626 via motor coupling 629. Motor 626 is mounted onsubstructure 624 that is fastened firmly to base enclosure 625. Anoperator activated latch 622 ensures that disposable portion 600 isfirmly located relative to reusable portion 601 by engaging in anannulus integral with drum 613.

Disposable portion 600 arrives as a sterile unit and is used adjacent toa sterile field in an operatory environment. On completion of theprocedure for preparing and applying PRP or PPP (which could involverunning the device multiple times for multiple applications for a singlepatient) disposable portion 600 is discarded into the bio waste stream.However the reusable portion 601 remains in the operatory and may getmoved elsewhere for storage. To ensure that no whole blood or bloodcomponents contaminate the reusable portion 601, a variety of elementsmay be employed to prevent the egress of these fluids. With reference toFIG. 26, absorbable washers 632 and 636 can capture any spillage fromreceiver 647, and gel accelerator 649 can cause the discharged fluids inreceiver 647 to gel into a non-flowing gelatinous mass. Alternativelysealed bearings (not shown) at 619 and rolling diaphragms (not shown)between drum 613 and follower 618 can capture all liquids. Absorbablematerials can be made from porous polyethylene (as sold under the tradename ‘Porex’), superabsorbent polymers, polyacrylates, polyelectrolytes,hydrogels, chalk, cellulose fibers or sponges, or woven textile, orother suitable materials known in the art. Gel accelerators can be madefrom materials as supplied by Multisorb Technologies, Inc. under thename Drimop®. Residuals of the PRP collected in the chamber arecontained by port valve 602. Combinations of these solutions to leakagewill also be clear to those skilled in the art.

FIGS. 28-31 depict a radial indexing valve receiver 700, which is analternate embodiment of the previously discussed RBC-plasma receiver647. This radial indexing valve receiver incorporates a radial indexingvalve that works in cooperation with the rotating drum 613 and follower618 (as shown previously in FIG. 21) in order to prevent the contentsfrom spilling from the receiver. The radial indexing valve receiver 700consists of two mating components, the upper valve 701 and the lowerstorage chamber 702. The upper valve 701 preferably includes four slots704 and the lower storage chamber 702 includes four slots 705. Thenumber of slots can be varied and typically the numbers of slots in eachcomponent are the same. The upper valve 701 includes indexing tabs 703that cooperate with grooves (not shown) in drum 613 so that the uppervalve 701 rotates when drum 613 is rotated. The upper valve 701 alsoincludes a 360 degree liquid inlet window 707. The lower storage chamber702 includes grooves 706 on its inner circumference that cooperate withtabs (not shown) on follower 618. Grooves 706 serve to key the lowerstorage chamber 702 to the follower 618 and prevent the lower storagechamber 702 from rotating when drum 613 is rotated. With reference toFIG. 30, the upper valve 701 and lower storage chamber 702 includeannular interlocking features 709 and 708. As can be seen in greaterdetail in the disassembled depiction of FIG. 31, the interlockingfeatures include the slots 704, 705 and mating surfaces 710, 711. As canbe seen in FIG. 30, the interlocking features 709, 708 define aninterference fit so that upper valve 701 and lower storage chamber 702can be snapped together where mating surfaces 710 and 711 create a watertight seal. In use, the receiver 700 is to be supplied in the positionas shown in FIG. 28 where the slots 704 in the upper valve 701 do notoverlap with slots 705 in the storage chamber 702. The centrifugechamber 646 is then to be filled with blood and the centrifuge isactivated. When drum 613 is rotated to open RBC valve port 638 (aspreviously discussed with reference to FIG. 21), the upper valve 701rotates with the drum, thus at least partially overlapping slots 704 and705 and thereby creating a passage between the two receiver components701 and 702, and as seen in FIG. 29. The expelled RBC's 641 enter theupper valve 701 through 360 degree liquid inlet window 707 and drain bygravity into lower storage chamber 702, through the overlapping regionof the slots. When drum 613 is rotated back to its home position to stopthe flow of RBC's 641 from valve port 638, slots 704 and 705 return tothe non-overlapping position shown in FIG. 28 thus sealing the RBC's inthe lower storage chamber 702. Similarly, when drum 613 is rotated inthe opposite direction to open the plasma port 612 the opposite sides ofslots 704 and 705 are caused to overlap, thus allowing the ejectedplasma 643 to drain into the lower storage chamber 702 through theoverlapping slots. Drum 613 is then rotated back to its home position atthe end of the process to return slots 704 and 705 to thenon-overlapping position, thus sealing the discarded fluid in the lowerstorage chamber 702. This prevents any spillage of the fluid duringsubsequent handling and disposal of the disposable portion 600.

Typical dimensioning of slots 704 and 705 is such that there will beoverlap when the upper valve 701 is rotated in either direction. In apreferred embodiment, the upper valve slots 704 each encompass 30degrees of the circumference while the lower storage chamber slots 705encompass 50 degrees of the circumference. This dimensioning leaves 5degrees between the edges of the slots, when in the closed orientation.Drum 613 is to be rotated approximately 35 degrees to open ports invalve cap 616. This will cause an overlap of slots 704 and 705 of 30degrees, or put another way, each entire slot 704 of the upper valve 701will be totally open to the lower storage chamber 702 through slot 705.Other combinations of slot geometry and placement are possible and wouldbe obvious to one skilled in the art. The upper valve 701 and storagechamber 702 are typically blow molded components, using resilientthermoplastic resins, including but not limited, to polypropylene andpolyethylene.

Reusable portion 601 is powered by a cord mounted transformer (notshown) from an AC supply, or from a DC power pack such as those used forcordless drills and the like. Additional items not shown are (but notlimited to) a simple display mounted on the base enclosure 625 thatindicates power on-off to the centrifuge, elapsed time from power on,and may include items such an audible alarm for warning the operatorwhen elapsed times reach certain levels. In addition hall-effectswitches or reed switches (not shown) mounted in the base 625 whichrespond to magnets mounted in the disposable portion 600 can be used toindicate the rotation of drum 613 in base enclosure 625, and-or can beused to select varying motor speeds that might be necessary for optimumseparation of fluid components.

Instead of an operator revolving drum 613 manually, actuators (e.g.motor-gearbox combinations or screw jacks) in the base 625 can rotatethe drum automatically in response to signals from the switchesdescribed above and-or from a small solid state computer employed tooptimize operation.

FIG. 22 is a simplified transverse section of FIG. 21 at AA. The bloodhas separated into its major components plasma 643, RBCs 641, andBuffy-coat (BC) at 642.

FIG. 23 is a simplified transverse section through BB of FIG. 21. Thissection shows the construction of the plasma valve consisting of passage610, and ‘O’ ring 611. The construction of these outlet ports is similarto that shown in FIG. 3b . When this valve is opened, port 612 will bemoved to a position in alignment with passage 610, to allow for the flowof fluid therethrough.

FIG. 24 shows the device of FIG. 21 running in the situation where theRBC valve port 638 is closed, the plasma port 612 is open, and theplasma discharge has been completed. The volume of plasma 643 is thefinal volume.

When platelet poor plasma (PPP) is required for a procedure, a slightlydifferent configuration is required for the PPP receiver. FIG. 25 hasmost components similar to those shown in FIG. 21 but there are tworeceivers, one for RBCs 637 and one for PPP 635. Since two fluidcomponents are captured by discharge from the spinning chamber thereceivers both have to be fixed axially relative to drum 613 to acceptthe different axial locations of the plasma port 612 and RBC port 638 asthey discharge appropriate fluid component. A plasma access port 645spans the walls of the receiver 635 and extends through slot or opening(not shown) in drum 613. This port is of elastomeric material such asnitrile rubber that permits the passage of a hypodermic needle for theremoval of the PPP.

In use the operator places a sterile disposable portion 600 into thereusable portion 601, the drum position being preset at the factory tothe position where both plasma port 612 and RBC port 638 are closed. Theoperator then fills a syringe with whole blood from the patient andintroduces the blood via the syringe through port valve 602 into thecentrifuge chamber until the chamber is filled. The device is activatedand the motor runs for about one minute by which time the blood hasseparated into the primary layers of RBC, buffy-coat, and plasma. Atthis time the drum is turned to position the RBC valve to the openposition whereupon RBCs start to discharge into receiver 637. As theRBCs discharge, the interface between RBCs and buffy coat (D5 in FIG.20a ) approaches markings on the rotating barrel at 644 (L5 and L4 ofFIG. 20a ). When the interface is between marks at 644 (about 30 secondsafter the RBC port 638 is opened) the drum is turned to close the RBCport and open the plasma port 612. Plasma then discharges into thereceiver and continues to do so until the air core limits furtherdischarge. At this point (about 30 seconds after the plasma port wasopened) the motor is stopped and the enriched residual sample in thechamber is removed via port 602 with a syringe and cannula for injectioninto the patient (or onto material about to be used as an implant). Inthe case of a PPP preparation the process is the same as that describedfor PRP except that the device conforms to the device shown in FIG. 25and the PPP is extracted from the side elastomeric port 645 of FIG. 25.

It is recognized that by employing varying speeds of centrifugation, andaltering the diameters at which the outlets from the chamber are placed,it is possible to concentrate different components, or isolate differentspecific gravity fractions of the fluid material within the rotationchamber. For example, rotating at a slower speed, as known to thoseskilled in the art, and removing the bulk of the RBCs as describedabove, will provide a plasma material with the suspended platelets. Whenrotated at lower speeds, the platelets will not differentiate byspecific gravity from the plasma. Upon increasing the speed of rotation,the platelets will then tend to differentiate by specific gravity fromthe plasma, allowing flexibility in achieving the desired combination ofblood products sought by the operator.

While the various embodiments discussed previously have described theblood separation chamber having a circular cross section, it isrecognized that any shape capable of high speed rotation can beutilized, so long as there is an angled or tapered inner diameter tofacilitate the appropriate flow of the red blood cells towards the RBCpassage. For example, a separation chamber that provides an ovalizedcross section may be employed, as it will be properly balanced andsuitable for the rotational speeds required. Similarly, other separationchambers having cross-sectional profiles in varying shapes (e.g.,octagonal, square, triangular, etc.) can be employed, and if necessary,balanced with weights to ensure proper balance when rotating.Furthermore, it is also recognized that multiple chambers may beutilized in the device, such as by providing 2 or more sections of acircle, or alternatively 2 or more vessels may be balanced to allowrotation of the multiple chambers, collectively forming a rotor, whereeach of the chambers would provide for discharge of particular bloodcomponents (e.g., RBC and Plasma), while allowing for the concentrationand access to the desired blood component concentrated in each of thechambers.

The embodiments described herein are chiefly intended for use inseparating components from whole blood, though they may be used withother liquids as well. In the case of blood product, once the device hasbeen operated to stratify the blood into its constituent components, andthe red blood cells and plasma removed from the blood separation chambervia the previously described RBC and plasma passages, the concentratedbuffy coat containing platelets and white blood cells will remain withinthe chamber. In all the embodiments discussed, the operator of thedevice may further choose to clarify the resulting buffy coat by addingone or more additional biocompatible solutions, as a separation aid,into the device and optionally performing further centrifugation steps.These additional biocompatible solutions are sometimes referred to asfocusing fluids. As previously described, the buffy coat consists ofseveral constituents, including platelets and leukocytes (i.e. whitecells), each having unique specific gravities. The leukocytes containgranulocytes and lymphoid cells such as lymphocytes and monocytes, eachof these having unique specific gravities. For some applications, it maybe important to isolate or remove one or several of these componentsfrom the buffy coat to provide a further purified therapeutic material.For instance, some researchers have found improved in vitro performanceby removing leukocytes from the buffy coat (S. R. Mrowiec et al., Anovel technique for preparing improved buffy coat platelet concentrates,Blood Cells, Molecules and Diseases (1995) 21 (3) February 15: 25-23).By way of example, a fixed quantity of one or more liquids (e.g.focusing fluids) having specifically targeted specific gravities couldbe delivered into the blood separation chamber to allow furtherseparation of various components of the buffy coat (e.g. leukocytes)thereby focusing in upon a very specific sub-component of the blood.Alternatively, a focusing fluid may be used to enable the removal of allof the red blood cells or plasma, by being of a targeted specificgravity between the buffy coat and either the red blood cells or theplasma components, such that by repeating the concentration processdescribed above, a blood component free from residual traces of eitherthe plasma or red blood cells may be achieved. Such focusing fluidscould include colorant, markers or other indicators to help distinguishthe boundaries between the targeted and non-targeted biologiccomponents. Fluids such as Ficoll-Paque sodium diatrizoate solution(density of 1.077 g/mL, distributed by GE Healthcare), Percoll (densityof 1.088 g/mL, distributed by GE Healthcare), and Cellotion (distributedby Zenoaq) and other fluids known in the art could be used forpurifying, separating and/or concentrating a wide variety oftherapeutically beneficial cells and other biological constituents.

In another embodiment the biocompatible focusing fluid may selectivelybind to a blood product and subsequently be isolated or separated bycentrifugation, to result in a more concentrated desired bloodcomponent. Various techniques are known in the art for accomplishing thebinding, for example, solid bead components of desired specific gravitymay be coated with antibodies and employed to selectively bind thefocusing fluid layer with the targeted blood component (or conversely,the blood component to be separated from the desired blood component).Alternatively, various techniques and reagents known to one skilled inthe art, using techniques known, for example, from separation chemistry(e.g., chromatography or absorption) may be employed (such as ionexchange resins as used in HPL C and FPLC methodologies). In theseembodiments, upon adding the focusing fluid to the blood separationchamber containing the previously concentrated blood product, andallowed an opportunity to bind, the desired blood product will be causedto separate from the unwanted blood product when the rotation isemployed to stratify the materials within the blood separation chamber.Removal of separated products can proceed through one or both of theoutlets as described previously. The binding of the focusing fluid inthis embodiment may be reversible using techniques known in the art,such that upon being harvested, the blood component may be unbound fromthe focusing fluid, and optionally subjected to another purificationprocedure to provide harvested blood product free of any focusing fluid.

As before, with an operator or sensor causing the actuation of the valvemechanism controlling the discharge of fluids from the chamber, adetectable interface would be beneficial in determining when to closeoutlet valves. For this reason, the focusing fluid is preferablydistinguishable in some manner at the interface with the othercomponents within the chamber, for example, by being distinguishable bycolor. Alternatively, prior to the centrifugation with the focusingfluid, a biocompatible, selective dye or marker material may be added todistinguish the fluids within the chamber, and create the interface thatis detectable by the operator or sensor. Thus, the selective coloringwould facilitate detection of an interface between the desiredcomponents, and those components sought to be removed from the bloodseparation chamber through one or both of the outlet ports.

In another embodiment, device 750 is configured as shown in FIG. 32 inorder to directly apply a selected fraction of the blood sample to abiologically compatible scaffold 751 via the spraying action from port752 of the spinning chamber. Examples of scaffolds include but are notlimited to purified collagen pads or powder, extracellular matrix sheetor powdered products, bone void fillers and resorbable or non-resorbablesynthetic meshes. In the embodiment shown in FIG. 32, the cam 621 hasfour positions, 1-4, with position #1 being the lowest position, insteadof the three previously described with regards to earlier embodiments.Valve cap 616 has additional BC port 752 and end cap 614 has additionalBC passage 755. Following the centrifugation procedure as previouslydescribed, follower 620 is rotated from the neutral position #3 toposition #4 on cam 621 to align port 638 with RBC passage 639, such thatthe red blood cells exit from port 638 into receiver 647. At theappropriate time, follower 620 is rotated to the #2 position on cam 621to align port 612 with passage 610, such that plasma exits from port 612into receiver 647. In the final step, follower 620 is rotated toposition #1 on cam 621 to align BC port 752 with passage 755 to allow BCto exit to a BC receiver 753. In the embodiment of FIG. 32, the BCreceiver 753 contains a scaffold 751. The scaffold 751 can receive theBC as it is discharged into the BC receiver; for example, the scaffoldmay be directly sprayed with the BC or alternatively, the BC can wickinto the material by capillary action or absorption into the scaffold751. When the desired amount of BC has exited at port 752, the follower620 is returned to neutral position #3 on cam 621 and the motor switchedoff. Scaffold 751, now treated with BC, may then be aseptically removedfrom receiver 753. While various access methods may be employed, oneexample is to provide access by disengaging mid-cover 607, at connectionjoint 754, to provide for scaffold access. Using analogous alterationsof the design of receivers, cam and ports, it is possible to directlyapply any of the blood fractions to a desired scaffold.

Direct application of BC to a scaffold results in a time savings, andless chance for contamination of the preparation, since the applicationis done automatically in a closed system rather than manually in an opencontainer. It would also reduce the chances for infection of health careworkers by reducing the amount of handling of a blood product thatpotentially contains a human pathogen.

In order to prevent premature destruction of the blood cells that arebeing applied to the scaffold 751 in receiver 753, the force at whichthe materials are ejected from the centrifugation chamber can becontrolled. By example, it has been shown that in a device with a 30 mlcapacity, with an exit hole diameter of 0.01″, the centrifuge gave ahigher proportion of intact cells when the centrifugal force (g) wasreduced to below about 1000 g, with additional improvement in cellsurvival as speeds were reduced further, to the range of about 300 g.Changes in centrifugation speed can be programmed in the software toautomatically initiate by timers in the software or signals generated bymovement of the valve mechanisms.

Another alternative, exemplary embodiment of a centrifuge 100constructed in accordance with this invention is shown in FIG. 34. Thatcentrifuge is also arranged for separation, concentration and collectionof select constituents of a biologic liquid mixture (such as, but notlimited to, blood) and basically comprises two main assemblies, namely,a removable disk-shaped rotating assembly 112 and a housing 113. Thehousing contains the rotating assembly, a drive motor 126 and associatedelectrical drive circuits (not shown) and switches (not shown). Theremovable disk-shaped rotating assembly 112 basically comprises aseparation chamber 143, and, in this exemplary case, two collectionchambers 109 and 110 for receipt of separated constituents. Inparticular, in the exemplary embodiment depicted, the rotating assemblycomprises a first or outer collection chamber 110 for the collection ofhigher specific gravity fluids, e.g., RBCs and a second or innercollection chamber 109 for the collection of lower specific gravityfluids, e.g., plasma. Each of these chambers is annular in shape, butcould be of a different shape, so long as the rotating assembly wasconstructed to be balanced to prevent unbalance-induced vibration. Acover 101 encloses the rotating assembly 112 when the centrifuge isrunning. In this embodiment, it is envisioned that the housing 113, withits various internal components would be reusable inasmuch as it wouldnot directly come into contact with the biologic liquid mixture. Theremovable rotating assembly 112, in contradistinction may be considereddisposable after use.

The three chambers of the rotating assembly 112 are made up from severalcomponents, including a main body 108 having an extending hub 127, acover plate 105, and a valve plate 115. These three components aresecured together to provide leak-proof seals at all the interfaces.Alternatively, these components may be of unitary construction andformed as a single piece, using various manufacturing techniques,including, for example, stereolithography or casting. The fixed valveplate 115 is fixedly secured to the extending hub 127, so that arotational force applied to the hub will result in the rotation of therotational assembly. The rotating assembly also features an access portin the form of a pierceable membrane 144 located in the cover plate 105to serve as the means for introduction of the biologic liquid mixtureinto the separation chamber 143, for processing. The access port can beof any suitable construction, e.g., a one-way duckbill valve. The accessport may also serve as the means for removal of any residual separatedcomponent of the biologic mixture, e.g., the buffy coat if the biologicmixture is blood.

In operation, fluid flow between the separation chamber 143, and thecollection chambers 110 and 109, is controlled by valves 138 and 117 (tobe described later) that can be actuated independently, to selectivelyopen and close, in order to control the flow of fluid therethrough. Thevalves are in fluid communication with the interior of the separationchamber via fluid passageways 141, 140 and 106, and will thus rotate aspart of the rotating assembly. The fluid passageway 140 constitutes achannel between the underside of the top plate 105 and the body 108 andis in fluid communication with the interior of the separation chamber143. The passageway 141 is in fluid communication with passageway 140.The passageway 140 is annular in shape and is in fluid communicationwith the valve 138 which is located at the passageway's terminus. Itshould be pointed out at this juncture that the passageway 140 need notbe of annular shape. If not annular in shape, the rotating assemblyshould include either a similarly shaped passageway diametricallyopposed to the passageway 140 or something else to balance the assemblyso that will not vibrate upon rotation. The fluid passageway 106 is alsoin fluid communication with the interior of the separation chamber 143,but at a smaller radial distance than the fluid passageway 141. Thepassageway 106 is in fluid communication with the valve 117, which islocated at the passageway's terminus. The passageway 106 is not annularin shape, but rather constitutes a bore. Preferably, a similarpassageway is located diametrically opposed to the passageway 106 toresult in a balanced assembly. If desired the passageway 106 could beannular.

As will be explained, the valves 138 and 117 feature elements that arestatic and dynamic relative to the other elements of the rotatingassembly. The static elements are fixed with respect to the rotatingassembly while the dynamic elements are arranged to shift or pivot,relative to the rotating assembly.

As shown in in FIG. 34, and in expanded detail in FIGS. 35 and 37, eachof the valves 138 and 117 is a 2-way (on/off) valve. Each valve includesa common valve plate 115 which forms the static (stationary) portion ofeach valve. The movable (slidable) portion of each valve is in the formof an arcuate section or segment of a common valve slider 116 (FIG. 37).Each valve is arranged so that when in its open position it permitsfluid flow through it into its respective collection chamber. Inparticular, when open, the valve 138 permits fluid to flow through itinto the collection chamber 110. Similarly, when open, the valve 117permits fluid to flow through it into the collection chamber 109. Thus,the valves enable the controlled transfer of separated constituents ofthe biologic liquid mixture from the separation chamber 143 to thecollection chambers 110 and 109.

To ensure leak proof operation, the valves employ resilient sealingmaterials between the respective parts of the valve that oppose eachother and slide or pivot relative to each other. In particular, asclearly shown in FIG. 35, which represents the construction of each ofthe valves 138 and 117, the static portion of each valve, i.e., thevalve plate 115, houses two O-rings 146 in respective circular recessesin the valve plate 115. Two pillars 147 and 148, which are integral withthe valve plate 115, project downward therefrom. The pillars cooperatewith a support guide cover 152 which is fixedly secured to each pillarto define a slot therebetween. The cross section of the slot isdesignated by “abcd” (see FIG. 35). The slot abcd of each valve extendsbelow the valve plate 115, parallel to the plane of the valve plate andis arranged to slidably receive therein a respective arcuate segment ofthe common valve slider 116. In particular, the slot of valve 138slidably receives the arcuate segment 154 (FIG. 37) of the valve slider116, while the slot of the valve 117 slidably receives the arcuatesegment 153 of the valve slider. The valve slider 116 is arranged to bepivoted about the central longitudinal axis (the rotation axis X shownin FIGS. 34 and 37) of the rotating assembly 112 to operate (i.e., openand close) the valves 138 and 117, as will be described later.

As best seen in FIG. 35, the valve plate 115 includes an entrance portor hole 182 and an exit port or hole 183 for each valve. The ports 182and 183 extend through the associated portion of the valve plate intofluid communication with the slot abcd located therebelow. Each valveincludes a seal plate 150 which is fixedly secured to the underside ofits associated arcuate segment 153 or 154 and is located within the slotabcd of that valve. The arcuate segment 154 of the valve slider 116includes a pair of drillings or holes 149 bridged by a transverse slot151. The two drillings 149 of the arcuate segment 154 are arranged to bebrought into alignment and fluid communication with the ports 182 and183 of the valve 138 when the valve slider 116 is pivoted in onerotational direction about the axis X. Similarly, the two drillings 149of the arcuate segment 153 are arranged to be brought into alignment andfluid communication with the ports 182 and 183 of the valve 117 when thevalve slider 116 is pivoted in the opposite rotational direction aboutthe axis X. Accordingly, when the valve slider 116 is pivoted to theposition aligning its drillings 149 with the ports 182 and 183 of thevalve 138 the higher specific gravity fluid that is separated by thecentrifugation is permitted to flow from the separation chamber 143through the channel 141 down the passageway 140 into port 182. Fromthere that fluid flows through the transverse slot 151 to the exit port183 from whence it flows into the chamber 110. When the valve slider 116is pivoted to the position aligning its drillings 149 with the ports 182and 183 of the valve 117 the lower specific gravity fluid that isseparated by the centrifugation is permitted to flow from the separationchamber 143 through the passageway 106 into port 182. From there thatfluid flows through the transverse slot 151 to the exit port 183 fromwhence it flows into the chamber 109.

The dimension of the slot abcd are selected to provide appropriatecompression to O-rings 146 to prevent leakage when the arcuate slidersegments 153 and 154 slide through to open and close the valves.

Since the valves 138 and 117 are part of the rotating assembly, a forcetranslating mechanism is provided to effect the actuation of the valves,while the device is in operation. This force translation may beaccomplished using various techniques known to those skilled in the art.One exemplary force transfer mechanism suitable for use in the devicewill now be described.

In order to enable the valve slider to be pivoted in the two rotationaldirections about axis X with respect to the valve plate 115, a pair ofcomponents with helical teeth are provided. Those components arearranged to be moved axially relative to each other as best seen in FIG.36, all while rotating to create a centrifugal field for the separationof the biologic liquid mixture. Referring to FIG. 34, it can be seenthat the chamber body 108 has male axial splines 121 incorporated on theextending hub 127. A translator, 122 having female splines slidesaxially on the male splines 121 of the extending hub 127. The translatorincludes helical teeth 120 on its exterior. A valve slider hub 184 hasinner helical teeth 118 that mate with the exterior teeth 120 on thetranslator. The translator 122 is moved axially via ball bearing 130 bythe axial motion of a sleeve 132. That sleeve is keyed with slots 137 inportions of housing 113. The sleeve 132 is driven axially in turn bycams 124 formed on the interior of a control sleeve 136. The controlsleeve 136 is arranged to be rotated, driven by tabs 134. The tabs passthrough slots in the housing 113. The control sleeve 136 has threepositions, with the middle position corresponding to the valve platepositions shown in FIG. 37. As the control sleeve 136 is rotated, thesleeve 132 riding on cams 124 creates an axial motion, which thetranslator 122 converts to a rotary motion, which in turn effects thepivoting of the valve slider 116, so as to direct the actuation of thevalves. A return spring (not shown) biases the valve plate 115 intorsion, such that the translator 122 is driven toward the motor 126.

The entire removable rotating assembly 112 mounts on the drive shaft 123of the motor 126, and is held in place by wing nut 103. The rotation ofthe rotating assembly 112 is driven by the motor 126 and an associateddriveshaft 123 through a key 104. The driveshaft 123 is located by ballbearing 145 and the motor drive end 125. The motor 126 is mounted on aframe 128 that also locates the ball bearing 145.

In operation of the device 100, the control sleeve 136 is placed in thecentral position, controlled by detents (not shown). A new removablerotating assembly 112 is placed over the drive shaft 123 and presseddown to compress the return spring (not shown) and thus position thevalve plate 115 in its starting position, that is with the two valveslots 151 in the position shown in FIG. 37. The wing nut 103 is thenspun onto threaded portion 102 of driveshaft 123, to hold the removablerotating assembly 112 in place. A charge of fluid (the biologic liquidmixture, e.g., blood) for separation is injected into chamber 143 viamembrane 144. The motor 126 is turned on and allowed to run at a speedappropriate for separation of the biologic liquid mixture into itsseveral constituent layers as concentric layers within the separationchamber 143. At adequate rotational speeds, this separation will occurin approximately ninety seconds or less, more typically about oneminute. For mixtures with very similar specific gravities, this timeperiod may be extended. Once separation of the constituents hasoccurred, the tab 134 is moved into a second position, to pivot thevalve slider 116 in one rotational direction about the axis X to theposition such that one of the holes 149 in the first valve 138 alignswith the entrance port 182 and the other of the holes 149 aligns withthe exit port 183, thereby opening that valve so that the high specificgravity fraction of the biologic liquid mixture flows into chamber 110through first valve 138.

In accordance with one preferred embodiment of this invention, theinterface created by the separation of the biologic liquid mixture intofractions is visually detectable, such that it may be observed through atransparent portion in the top plate 105. Alternatively, the interfacemay be detected electronically, using sensors as has been discussedpreviously. It is recognized that there may be a benefit in providing acontrasting color, or mirrored surface, over at least a portion of therotating chamber disposed opposite a detector, or opposite to thetransparent portion of the top plate of the rotating assembly, in orderto enhance the detectability of the interface by the operator ordetector.

The movement of the interface is monitored as the higher specificgravity constituent flows into chamber 110. As this interface approachesthe entrance 142 to channel 141, and when the interface reaches apredetermined point, relative to the entrance 142 into channel 141, thefluid flow through first valve 138 is halted, such as by acting upon thetab 134 to trigger the closing of the first valve 138. Though the timefor the evacuation of the higher specific gravity fluid will vary basedupon the speed of the rotation, the viscosity of the fluid, and thediameter of the most restrictive portion of the flow-path, it isanticipated that this will typically be after approximately 15 secondsof flow. Where the operator is detecting the interface, and monitoringin order to determine when to close the first valve, it is necessarythat the rate of movement of the interface is such that a person mayhave adequate time to react and trigger the closing of the valve, as hasbeen discussed previously.

Subsequently, the tab 134 is then moved to a third position, which thenpivots the valve slider 116 in the opposite rotational direction aboutaxis X to the position such that one of the holes 149 in the secondvalve 117 aligns with the entrance port 182 and the other of the holes149 aligns with the exit port 183, thereby opening that valve so thatlower specific gravity fraction of the biologic liquid mixture flowsinto chamber 109 through the second valve 117. This flow continues untilair enters passageway 106, whereupon flow ceases. At this juncture,typically about 2-3 minutes into the procedure, the tab 134 is returnedto its original, first position, with both valves 138 and 117 nowclosed, and the motor is de-energized. A predetermined volume of thelower specific gravity constituent, together with the remains of anyintermediate specific gravity constituents (e.g., buffy coat, if thebiologic mixture is blood), are now trapped in the separation chamber143, and ready for removal for use, for example, by directing a syringeinto the separation chamber via membrane 144. The removable rotatingassembly 112 may then be separated from the driveshaft 123, and thecontents of one or more of the three chambers 143, 108, and 109 can beharvested if desired (via membrane and syringe, for example).

As will be appreciated by those skilled in the art, in order to functionproperly, the device 100 incorporates one or more vents (not shown) toallow for fluid displacement. Ideally, one of the vents provides for airto enter the chamber 143 as the fluid flows into chambers 109 and 110,and another two vents allow air to escape as fluid flows into chambers109 and 110.

In an embodiment of the centrifuge 100, it may be desirable to providefor the separation of a biologic liquid mixture, e.g., blood, charge ofapproximately 30 mL. To that end the above described rotating assembly112 may be sized with a maximum diameter of approximately 8 cm, and aheight measured along the longitudinal axis (the rotation axis X) ofapproximately 1 cm. It is recognized that increasing or decreasing thedimensions may be desirable in order to achieve a desired sample sizefor processing.

As has been discussed previously, it is recognized that the chambers ofthe device depicted in FIGS. 34-37, may be either annular chambers, oralternatively, may be a plurality of linked chambers forming two or moresections of a circle, or alternatively, two or more vessels may bebalanced to allow rotation of the multiple chambers, collectivelyforming a rotor, where each of the chambers provides for discharge ofparticular blood components (e.g., RBC and Plasma), while allowing forthe concentration and access to the desired blood component concentratedin each of the chambers.

In any of the embodiments described herein, the device may benefit fromthe incorporation of a tilt sensor that would halt the rotation, or at aminimum reduce the rotation speed, of the chamber, should the device betoppled or operated at an undesirable angle. Given the rotation speedthat the device is expected to operate under, the angle would not likelyaffect the stratification of the fluid components, but rather, thiswould prevent unintended movement, that is known with devices thatinclude rotating elements, or those prone to vibrate while in operation.

The above described embodiments may be made available in kit form,including the device and accessories needed for operation of the device,including instructions for use and packaging suitable for storage andpreserving sterility. In some instances, the kit may provideinstructions along with the centrifuge device (either as a single unit,or separable components), and optionally including accessories such asseparation aids, including focusing fluids, or rapid test kits usefulfor providing qualitative or quantitative information regarding theconcentrated product. Various blood testing procedures are known in theart, but it is anticipated that any rapid testing kit that providesuseful information regarding, for example, the concentration factor orrecovery efficiency of the concentrated products may be incorporatedinto the kit. Such a kit may require comparing the results for bloodcomponents that have been subjected to the rapid test kit afterconcentration, and optionally prior to concentration. It is envisionedthat the accessories may be contained within a separate container withinthe packaging, or contained within the blood separation chamber duringpackaging, or made available apart from the centrifuge unit. For theembodiment providing a reusable drive component with a motor that isarranged to be coupled to a disposable centrifuge component, the kit mayinclude multiple disposable centrifuge components each suitable for usewith the reusable drive component.

Thus since the inventive process and inventions disclosed herein may beembodied by additional steps or other specific forms without departingfrom the spirit of general characteristics thereof, some of which stepsand forms have been indicated, the embodiments described herein are tobe considered in all respects illustrative and not restrictive. Thescope of the invention is to be indicated by the appended claims, ratherthan the foregoing description, and all changes which come within themeaning and range of equivalency of the claims are intended to beembraced therein.

What is claimed is:
 1. A method of isolating and concentrating afraction of a sample comprising bone marrow aspirate, the methodcomprising the steps of: a. providing a centrifuge comprising: i. achamber comprising a barrel having a longitudinal axis, a first end, asecond end, and a sidewall extending between said second end and saidfirst end, wherein said first end has a diameter smaller than thediameter of said second end, ii. a first port provided in said chamberat a first radial distance from said longitudinal axis, and in fluidcommunication with a first valve by a first passageway, iii. a secondport provided in said chamber at a second radial distance from saidlongitudinal axis and in fluid communication with a second valve by asecond passageway, with said second radial distance being less than saidfirst radial distance, iv. a motor to rotate said chamber about thelongitudinal axis; b. introducing a sample into the chamber, wherein thesample comprises bone marrow aspirate and wherein the sample comprisesconstituents having at least two different specific gravities; c.rotating said chamber about said longitudinal axis, and separating saidsample by specific gravity into a first fraction and a second fraction;d. selectively opening said first valve to eject at least a portion ofsaid first fraction from said chamber through said first port, therebyleaving a residual of said sample in said chamber; and e. selectivelyopening said second valve after the opening of said first valve, toeject at least a portion of said residual of said sample from saidchamber through said second port.
 2. The method of claim 1, wherein saidsample further comprises blood.
 3. The method of claim 1, furthercomprising the step of: f. collecting at least a portion of said sampleremaining within said chamber.
 4. The method of claim 1, furthercomprising between step d and step e, the step of monitoring aninterface between said first fraction and said second fraction throughsaid sidewall, and wherein at least a portion of the sidewall istransparent.
 5. The method of claim 1, further comprising between step dand step e, the step of detecting an interface occurring betweenseparated constituents of said sample by at least one automaticdetector.
 6. The method of claim 1, further comprising between step dand step e, the step of closing said first valve.
 7. The method of claim4, further comprising between step d and step e, the step of closingsaid first valve.
 8. The method of claim 5, further comprising betweenstep d and step e, the step of closing said first valve.
 9. The methodof claim 1, wherein at least a portion of the chamber is transparent,thereby permitting visualization of at least a portion of the sample.10. The method of claim 1, wherein the side wall extends in a taper fromthe second end to the first end.
 11. The method of claim 10, whereinsaid side wall extends in a uniform taper from said second end to saidfirst end.
 12. The method of claim 1, wherein the centrifuge furthercomprises an end cap for selectively closing the second end of thebarrel.
 13. The method of claim 12, wherein said chamber furthercomprises an annular wedge located within said barrel and projectingfrom said end cap, said annular wedge having a wedge side wall locatedadjacent said sidewall of said barrel to define therebetween acircumferential channel.
 14. The method of claim 12, wherein the firstpassageway and the second passageway extend through the end cap to theexterior of the chamber.
 15. The method of claim 14, wherein thecentrifuge further comprises a valve cap that is slidably fit over theend cap, said valve cap having a first outlet port and a second outletport, wherein said end cap and valve cap are movable relative to eachother, such that for said first valve, said first passageway can bebrought into alignment with said first outlet port in said valve cap inorder to selectively enable the flow of a fluid therethrough, and forsaid second valve, said second passageway can be brought into alignmentwith said second outlet port in said valve cap in order to selectivelyenable the flow of a fluid therethrough.
 16. The method of claim 15,wherein the movement of said end cap and valve cap relative to eachother is responsive to a signal.
 17. The method of claim 13, whereinsaid circumferential channel comprises a restrictive feature thatrestricts the flow of fluid in the circumferential channel.
 18. Themethod of claim 1, wherein said first passageway comprises a plenum. 19.The method of claim 1, wherein said second passageway comprises aplenum.